EP3134728B1 - Optogenetische analyse von verbindungen - Google Patents

Optogenetische analyse von verbindungen Download PDF

Info

Publication number
EP3134728B1
EP3134728B1 EP15720856.2A EP15720856A EP3134728B1 EP 3134728 B1 EP3134728 B1 EP 3134728B1 EP 15720856 A EP15720856 A EP 15720856A EP 3134728 B1 EP3134728 B1 EP 3134728B1
Authority
EP
European Patent Office
Prior art keywords
cells
optical
cell
neurons
reporter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP15720856.2A
Other languages
English (en)
French (fr)
Other versions
EP3134728A1 (de
Inventor
Kevin C. Eggan
Adam Cohen
Joel KRALJ
Evangelos Kiskinis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Q-State Biosciences Inc
Q State Biosciences Inc
Original Assignee
Q-State Biosciences Inc
Q State Biosciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Q-State Biosciences Inc, Q State Biosciences Inc filed Critical Q-State Biosciences Inc
Publication of EP3134728A1 publication Critical patent/EP3134728A1/de
Application granted granted Critical
Publication of EP3134728B1 publication Critical patent/EP3134728B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • C12N2502/081—Coculture with; Conditioned medium produced by cells of the nervous system neurons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2842—Pain, e.g. neuropathic pain, psychogenic pain
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/302—Schizophrenia

Definitions

  • the invention relates to methods of optically screening compounds for epilepsy treatment.
  • Epilepsy is a group of neurological disorders exhibiting a common symptom of epileptic seizures.
  • Epileptic seizures occur when a group of neurons begin abnormally synchronized firing causing a paroxysmal depolarizing shift.
  • Epileptic seizures may occur suddenly and without warning and may include the loss of consciousness and various degrees of muscle contractions or spasms followed by a period of confusion which may last for hours.
  • Individuals are also prone to physical injury during epileptic seizures. Additional symptoms include headaches, difficulty speaking, and psychosis. Beyond the physical effects, people suffering from epilepsy may suffer social restrictions such as being unable to drive or hold certain jobs due to the risk of seizure.
  • epilepsy In most cases, the cause of epilepsy is unknown but known causes include brain injuries associated with trauma, tumors, substance abuse, or stroke. Certain forms of epilepsy are linked to certain genotypes and mutations. Dravet syndrome, for example, is linked to mutations in the SCN1A gene. Understanding the mechanisms behind epileptic seizures and studying the neuronal effects leading up to, during, and after a seizure are key areas of focus.
  • Ion channels are important proteins in neurons and cardiac tissue as they are essential to the action potentials that make up our thoughts, sensations, and heartbeats. Those ion channels are thus significant therapeutic targets and many drugs function as ion channel modulators. Many ion channel modulators have been developed empirically by traditional pharmacology without knowing the precise target of those modulators. The discovery of novel ion channel modulators by high-throughput methods has proven challenging. A particular challenge has been the development of biologically relevant assays useful for screening sizeable compound libraries. Existing screening formats have limited throughput and do not provide the precision, temporal resolution, or voltage control needed for monitoring channel modulation.
  • the invention provides methods for screening compounds in high-throughput cellular assays of cells expressing optogenetic proteins that initiate and report electrical activity in cells using light.
  • the invention provides high-capacity methods for primary screening of chemical libraries.
  • These high-throughput assays provide robust electrophysiological measurements of cells without requiring patch clamp techniques. Since the described optogenetic constructs and pluripotent stem cell (PSC)-derived cells operate to provide the precision, temporal resolution, and voltage control required for monitoring effects of compounds, assays of the invention are compatible with primary screening and drug discovery.
  • a target protein may be cloned and expressed in a stable cell line of the invention.
  • the invention provides robust, biologically relevant assays with sufficient capacity for high throughput screening of compounds.
  • aspects of the invention provide a method for determining an effect of a compound a neurological condition.
  • the method includes presenting a compound to a sample comprising a plurality of neurons, wherein at least one of the plurality of neurons expresses an optical reporter of membrane electrical potential, and receiving-via a microscopy system-an optical signal generated by the optical reporter in response to optical stimulation of a light gated ion channel in the sample following presentation of said compound.
  • the compound is identified as a candidate for treatment of the neurological condition based on said optical signal.
  • the light gated ion channel may include an algal channelrhodopsin being expressed by a second neuron in synaptic communication with the at least one of the plurality of neurons.
  • the light gated ion channel may include an algal channelrhodopsin being expressed by the at least one of the plurality of neurons.
  • the optical reporter of membrane potential may include a microbial rhodopsin (e.g., with between 1 and 10 amino acid substitutions relative to a wild type form of the microbial rhodopsin).
  • the at least one of the plurality of neurons also expresses a genetically-encoded indicator of intracellular calcium level.
  • the received optical signal may include a signal from the genetically-encoded indicator of intracellular calcium level.
  • the neurological condition is epilepsy.
  • the invention offers disease models for in-vitro compound screening and study of Dravet syndrome and other genetic based neurological disorders associated with epilepsy by using neurons exhibiting a genotypic or phenotypic characteristic of Dravet syndrome or other forms of epilepsy.
  • Neurons for use in the invention are engineered to express an optical reporter of membrane electrical potential, a light-gated ion channel, and an indicator of intracellular calcium levels.
  • Neuronal cells for use in the invention are obtained from a number of different sources. For example, neuronal cells may be obtained from an individual having a neurological disorder, such as epilepsy. Cells may be obtained from a living donor or from postmortem tissue.
  • neuronal cells may be obtained from a cell bank, such as the American Type Culture Collection (ATCC) or other suitable source.
  • ATCC American Type Culture Collection
  • Neuronal cells having an epilepsy or Dravett syndrome phenotype or genotype may also be obtained through differentiation of a pluripotent stem cell using known methods.
  • Pluripotent stem cells may be human induced pluripotent stem cells (hiPSC) derived from somatic cells.
  • Disease genotypes or phenotypes may also be introduced into a neuron through genome editing.
  • action potentials may be optically induced and optically evaluated in vitro.
  • the neurons express a protein that reports a change in intracellular calcium level. The ability to optically obtain and observe action potentials and to observe changes in intracellular calcium level allows researchers to screen therapeutic compounds for epilepsy treatment.
  • Neuronal cells are transformed with a genetically-encoded optical reporter, such as a transmembrane protein that fluoresces in response to the generation of an action potential.
  • the optical reporter exhibits an optical signature as an action potential propagates through the neuron in response to neural stimulation (which may itself be optically induced).
  • the signature may be observed and compared to a control signature, such as may be observed from a control cell with known properties.
  • Neurons of the invention are used to screen potential compounds for therapeutic use.
  • Neuronal function e.g., action potential generation and propagation
  • Compound screening using transformed neurons may be used to evaluate the effectiveness of compounds or other treatments on preventing disease onset or progression or relieving disease symptoms.
  • Cell models for compound screening or other investigation may include a cell or cells which exhibit a phenotypic characteristic of a disorder such as Dravett syndrome or other forms of epilepsy.
  • Phenotypic characteristics may include, for example, a cell's morphological, biological, biochemical, electrochemical, or physiological properties.
  • Genotypic characteristics can include one or more mutations to an epilepsy-linked gene.
  • aspects of the invention use methods of converting stem cells to specific neural subtypes as well as transformation of cells with optogenetic actuators and reporters to enable optical characterization of cells. Images may be captured by microscopy and analyzed digitally to identify optical signatures, such as spike trains, and associate the signatures with specific cells. Disease-affected and healthy patient cells are distinguished according to their signature spike trains.
  • the cell is caused to express an optical actuator that initiates an action potential in response to optical stimulation.
  • Stimulation of the cell may include illuminating the optical actuator.
  • Causing the cell to express the optical reporter may be accomplished by transforming the cell with a vector bearing a genetically encoded fluorescent voltage reporter.
  • the vector may also include a genetically encoded optical voltage actuator, such as a light-gated ion channel.
  • Observing the signal can include observing a cluster of different cells with a microscope and using a computer to isolate the signal generated by the optical reporter from a plurality of signals from the different cells.
  • Methods of the invention may include using a computer to isolate a signal by performing an independent component analysis or other source-separation algorithm.
  • the computer may be used to identify a spike train associated with the cell using standard spike-finding algorithms that apply steps of filtering the data and then applying a threshold.
  • the computer may also be used to map propagation of electrical spikes within a single cell by means of an analytical algorithm such as a sub-Nyquist action potential timing algorithm.
  • Methods may include observing and analyzing a difference between the observed signal and the expected signal.
  • the difference may manifest as a decreased or increased probability of a voltage spike in response to the stimulation of the cell relative to a control, a change in the propagation of the signal within a cell, a change in the transformation of the signal upon synaptic transmission, or a change in the waveform of the action potential.
  • the invention provides methods for screening a compound for epilepsy treatment.
  • the methods include the steps of presenting a compound to a sample including a neuron with one or more phenotypic or genotypic characteristics of epilepsy and the neuron expresses an optical reporter of membrane electrical potential and a light-gated ion channel.
  • Methods of the invention include receiving, via a microscopy system, an optical signal generated by the optical reporter in response to optical stimulation of the sample following presentation of said compound, and identifying the compound as a candidate for epilepsy treatment based on the optical signal.
  • the phenotypic characteristic includes diminished voltage-gated sodium channel function compared to disease-free neurons and hyperexcitability.
  • the genotypic characteristic includes a mutation in a gene selected from SCN1A, WWOX, PRRT2, KCNC1, STX1B, CARS2, STXB1, KCNQ2, CDKL5, ARX, SPTAN, BRAT1 , KCNQ3, SCN2A ( NAV1.2 ), GABA receptors, NIPA2, CDKL5, PCDH19, and NAV1.1.
  • the microscopy system comprises a digital micromirror device that provides the optical stimulation.
  • the microscopy system may include a charge-coupled device camera configured to capture the optical signal from the neuron.
  • the neuron also expresses a protein that reports a change in an intracellular calcium level and is stimulated by a second neuron that expresses the light-gated ion channel.
  • the second neuron also expresses the optical reporter of membrane electrical potential.
  • the light-gated ion channel includes an algal channelrhodopsin and the protein that reports changes in intracellular calcium levels includes a GCaMP variant.
  • the protein that reports a change in an intracellular calcium level may be selected from the group consisting of jRCaMP1a, jRGECO1a and RCaMP2.
  • the neuron may be an hiPSC-derived neuron.
  • the method of the invention include the steps of detecting a change in the AP waveform and a change in the intracellular calcium level upon exposure of the neuron to the compound.
  • the method may include spatially patterning a plurality of neurons in the cell culture on a substrate.
  • the compound may be lacosamide or levetiracetam.
  • the identifying step may include comparing the optical signal of the sample to an optical signal obtained from a control cell.
  • the optical reporter of membrane electrical potential comprises a microbial rhodopsin which may include QuasAr! or QuasAr2.
  • the microbial rhodopsin can be expressed from a gene that is integrated into the neuron.
  • the light-gated ion channel may be a blue-shifted actuator and the blue-shifted actuator may include TsChR or PsChR.
  • the light-gated ion channel may include a blue-shifted actuator with an excitation maximum at a wavelength ⁇ 450 nm and the protein that reports the change in the intracellular calcium level can include a red-shifted calcium indicator with an excitation maximum between 520 nm and 570 nm inclusive.
  • Alzheimer's disease is a neurodegenerative disease of uncertain cause (although mutations in certain genes have been linked to the disorder) and is one of the most common forms of dementia.
  • Alzheimer's disease is discussed in Israel et al., 2012, Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells, Nature 482(7384):216-20 ; Muratore et al., 2014, The familial Alzheimer's disease APPV717I mutation alters APP processing and tau expression in iPSC-derived neurons, Human Molecular Genetics , in press; Kondo et al., 2013, Modeling Alzheimer's disease with iPSCs reveals stress phenotypes associated with intracellular Abeta and differential drug responsiveness, Cell Stem Cell 12(4):487-496 ; and Shi et al., 2012, A human stem cell model of early Alzheimer's disease pathology in Down syndrome, Sci Transl Med 4(124):124ra129 .
  • stem cell technology provides a clinically-relevant cell models of Alzheimer's and the use of microbial optogenetic constructs allows for rapid screening or detection of cellular physiologies and phenotypes.
  • FIG. 1 diagrams a method 101 for evaluating a condition according to aspects of the disclosure . This may involve obtaining 107 a cell (e.g., purchasing PSCs and converting to neurons; biopsy from a person suspected of having the condition; etc.).
  • a cell e.g., purchasing PSCs and converting to neurons; biopsy from a person suspected of having the condition; etc.
  • Genome editing techniques may be used to create a control cell that is isogenic but-for a variant of interest.
  • the cell and the control are converted into an electrically excitable cell such as a neuron.
  • the cell may be converted to a specific neural subtype (e.g., motor neuron).
  • the cells are caused to express 113 an optical reporter of neural activity.
  • the cell may be transformed with a vector comprising an optogenetic reporter and the cell may also be caused to express an optogenetic actuator (aka activator) by transformation.
  • a control cell may be obtained, e.g., by taking another sample, by genome editing, or by other suitable techniques. Using microscopy and analytical methods described herein, the cells are observed and specifically, the cells' response to stimulation 119 (e.g., optical, synaptic, chemical, or electrical actuation) may be observed. A cell's characteristic signature such as a neural response as revealed by a spike train may be observed 123. The observed signature is compared to a control signature and a difference (or match) between the observed signature and the control signature corresponds to a positive diagnosis of the condition.
  • stimulation 119 e.g., optical, synaptic, chemical, or electrical actuation
  • a cell's characteristic signature such as a neural response as revealed by a spike train may be observed 123. The observed signature is compared to a control signature and a difference (or match) between the observed signature and the control signature corresponds to a positive diagnosis of the condition.
  • Ion channels are typically multimeric, transmembrane proteins having separate pore-forming and accessory subunits ( Ashcroft, 2006, Nature 440:440-7 ). Ion channels are often classified according to gating mechanism: voltage-gated channels are regulated by changes in the electrical potential difference in membrane potential whereas ligand- and sensory-gated channels respond to changes ligands and to mechanical or thermal stimuli, respectively.
  • High throughput screening of large chemical libraries generally may include cloning of the target protein which is abundantly expressed in a stable cell line in a form that closely resembles its native correlates. For ion channels this involves efficient expression, localization, and orientation of an appropriate combination of subunits.
  • FIG. 1 diagrams a method 101 for evaluating a condition according to embodiments of the disclosure. This may involve obtaining 107 a cell (e.g., by converting a stem cell to a neuron). Genome editing techniques (e.g., use of transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system, zinc finger domains) may be used to create a control cell that is isogenic but-for a variant of interest. The cell and the control are converted into an electrically excitable cell such as a neuron or astrocyte. The cell may be converted to a specific neural subtype (e.g., motor neuron). The cells are caused to express 113 an optical reporter of neural activity.
  • TALENs transcription activator-like effector nucleases
  • the cells are caused to express 113 an optical reporter of neural activity.
  • the cell may be transformed with a vector comprising an optogenetic reporter and the cell may also be caused to express an optogenetic actuator (aka activator) by transformation.
  • a control cell may be obtained, e.g., by taking another sample, by genome editing, or by other suitable techniques. Using microscopy and analytical methods described herein, the cells are observed and specifically, the cells' response to stimulation 119 (e.g., optical, synaptic, chemical, or electrical actuation) may be observed. A cell's characteristic signature such as a neural response as revealed by a spike train may be observed 123. The observed signature is compared to a control signature and a difference (or match) between the observed signature and the control signature characterizes the cell.
  • stimulation 119 e.g., optical, synaptic, chemical, or electrical actuation
  • Cells may be obtained as stem cells (e.g., by purchasing for example iCells). Alternatively or additionally, cells are obtained from a person suspected of having a condition, e.g., as fibroblasts. Fibroblasts may be converted directly to neurons or may be converted to stem cells. Stem cells may be converted to neurons (e.g., by being forced to express a single transcription factor such as NgN2).
  • SHANK3 ProSAP2
  • CDH9 CDH10
  • MAPK3, SERT SLC6A4
  • CACNA1G CABRB3, GABRA4, EN2
  • SLC25A12 the 3q25-27 locus
  • HOXA1, HOXA2, PRKCB1, MECP2, UBE3A, NLGN3, MET, CNTNAP2, FOXP2, GSTP1, PRL, PRLR, and OXTR Genes such as the SHANK3 have been studied in mouse models through N-terminal and PDZ domain knock-outs which resulted in phenotypes including impaired social interaction.
  • Epilepsy is a genetic disorder suitable for analysis by a pipeline defined by methods of the invention.
  • Dravet syndrome also known as Severe Myoclonic Epilepsy of Infancy (SMEI)
  • SMEI Severe Myoclonic Epilepsy of Infancy
  • GEFS+ generalized epilepsy with febrile seizures plus (GEFS+) which is thought to include Dravet syndrome, borderline severe myoclonic epilepsy of infancy (SMEB), and intractable epilepsy of childhood (IEC).
  • Additional neurodevelopmental disorders associated with epilepsy which may be studied with the cells and methods of the invention include Angelman syndrome, Rolandic epilepsy, autosomal dominant nocturnal frontal lobe epilepsy, benign occipital epilepsies of childhood, Panalyiotopoulos syndrome, childhood absence epilepsy, epilepsy-intellectual disability in females, febrile lobe epilepsy, juvenile myoclonic epilepsy, Lennox-Gastaut syndrome, Ohtahara syndrome, photosensitive epilepsy, pyridoxine-dependent epilepsy, Unverricht-Lundborg disease, myoclonic epilepsy with ragged red fibers syndrome, Lafora disease, Rasmussen's encephalitis, ring chromosome 20 syndrome, temporal lobe epilepsy, tuberous sclerosis, and West syndrome.
  • Additional genes associated with epilepsy include, WWOX, PRRT2, KCNC1, STX1B, CARS2, STXB1, KCNQ2, CDKL5, ARX, SPTAN, BRAT1, KCNQ3, SCN2A (NAV1.2), GABA receptors, NIPA2, CDKL5, PCDH19, and NAV1.1.
  • fibroblasts may be taken from a patient known or suspected to have a mutation such as a mutation in SHANKS. Any suitable cell may be obtained and any suitable method of obtaining a sample may be used.
  • a dermal biopsy is performed to obtain dermal fibroblasts.
  • the patient's skin may be cleaned and given an injection of local anesthetic. Once the skin is completely anesthetized, a sterile 3 mm punch is used. The clinician may apply pressure and use a "drilling" motion until the punch has pierced the epidermis. The punch will core a 3 mm cylinder of skin.
  • the clinician may use forceps to lift the dermis of the cored skin and a scalpel to cut the core free.
  • the biopsy sample may be transferred to a sterile BME fibroblast medium after optional washing with PBS and evaporation of the PBS.
  • the biopsy site on the patient is dressed (e.g., with an adhesive bandage). Suitable methods and devices for obtaining the cells are discussed in U.S. Pat. 8,603,809 ; U.S. Pat. 8,403,160 ; U.S. Pat. 5,591,444 ; U.S. Pub. 2012/0264623 ; and U.S. Pub. 2012/0214236 .
  • tissue culture technique that is suitable for the obtaining and propagating biopsy specimens may be used such as those discussed in Freshney, Ed., 1986, Animal Cell Culture: A Practical Approach, IRL Press, Oxford Engl and; and Freshney, Ed., 1987, Culture of Animal Cells: A Manual of Basic Techniques, Alan R. Liss & Co., New York .
  • Obtained cells may be converted into any electrically excitable cells such as neurons, specific neuronal subtypes, astrocytes or other glia, or immune cells. Additionally, cells may be converted and grown into co-cultures of multiple cell types (e.g. neurons + glia, neurons + immune cells).
  • FIG. 2 illustrates exemplary pathways for converting cells into specific neural subtypes.
  • a cell may be converted to a specific neural subtype (e.g., motor neuron).
  • Suitable methods and pathways for the conversion of cells include pathway 209, conversion from somatic cells to induced pluripotent stem cells (iPSCs) and conversion of iPSCs to specific cell types, or pathways 211 direct conversion of cells in specific cell types.
  • iPSCs induced pluripotent stem cells
  • pathways 211 direct conversion of cells in specific cell types.
  • somatic cells may be reprogrammed into induced pluripotent stem cells (iPSCs) using known methods such as the use of defined transcription factors.
  • iPSCs are characterized by their ability to proliferate indefinitely in culture while preserving their developmental potential to differentiate into derivatives of all three embryonic germ layers.
  • fibroblasts are converted to iPSC by methods such as those discussed in Takahashi and Yamanaka, 2006, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Cell 126:663-676 .; and Takahashi, et al., 2007, Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell 131:861-872 .
  • Induction of pluripotent stem cells from adult fibroblasts can be done by methods that include introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions.
  • Human dermal fibroblasts (HDF) are obtained.
  • a retroviruses containing human Oct3/4, Sox2, Klf4, and c-Myc is introduced into the HDF.
  • the cells are harvested by trypsinization and plated onto mitomycin C-treated SNL feeder cells. See, e.g., McMahon and Bradley, 1990, Cell 62:1073-1085 .
  • DMEM containing 10% FBS fetal calf serum
  • bFGF basic fibroblast growth factor
  • iPS cells can then be differentiated into specific neuronal subtypes.
  • Pluripotent cells such as iPS cells are by definition capable of differentiating into cell types characteristic of different embryonic germ layers.
  • a property of both embryonic stem cells human iPS cells is their ability, when plated in suspension culture, to form embryoid bodies (EBs).
  • EBs formed from iPS cells are treated with two small molecules: an agonist of the sonic hedgehog (SHH) signaling pathway and retinoic acid (RA).
  • SHH sonic hedgehog
  • RA retinoic acid
  • Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons, Science 321(5893):1218-21 ; Amoroso et al., 2013, Accelerated high-yield generation of limb-innervating motor neurons from human stem cells, J Neurosci 33(2):574-86 ; and Boulting et al., 2011, A functionally characterized test set of human induced pluripotent stem cells, Nat Biotech 29(3):279-286 .
  • aspects of the disclosure provide cellular disease models in which stem cells may be converted into functional neurons by forced expression of a single transcription factor and then also caused to express optogenetic reporters or actuators of neural activity.
  • a transcription factor such as neurogenin-2 (NgN2) or NeurD1 introduced into a pluripotent stem cell by transfection is expressed, causing the cell to differentiate into a neuron.
  • an optogenetic construct that includes an optical reporter of intracellular calcium as well as an optical actuator or reporter of membrane potential is expressed.
  • conversion includes causing a stem cell to express a single transcription factor.
  • a single transcription factor such as neurogenin-2 (Ngn2) or NeuroD1 alone rapidly converts ES and iPS cells into neuronal cells.
  • the transcription factor may be introduced by lentiviral infection (discussed in greater detail below).
  • a puromycin resistance gene may be co-expressed with Ngn2 for selection.
  • ES or iPS cells are plated on day -2, infected with lentiviruses on day -1, and Ngn2 expression is induced on day 0.
  • a 24 hr puromycin selection period is started on day 1, and mouse glia (primarily astrocytes) are added on day 2 to enhance synapse formation.
  • Forced Ngn2 expression converts ES and iPS cells into neuron-like cells in less than one week, and produces an apparently mature neuronal morphology in less than two weeks, as reported in Zhang 2013.
  • pathway 211 human somatic cells are obtained and direct lineage conversion of the somatic cells into motor neurons may be performed. Conversion may include the use of lineage-specific transcription factors to induce the conversion of specific cell types from unrelated somatic cells. See, e.g., Davis-Dusenbery et al., 2014, How to make spinal motor neurons, Development 141:491 ; Graf, 2011, Historical origins of transdifferentiation and reprogramming, Cell Stem Cell 9:504-516 . It has been shown that a set of neural lineage-specific transcription factors, or BAM factors, causes the conversion of fibroblasts into induced neuronal(iN) cells. Vierbuchen 2010 Nature 463:1035 .
  • MicroRNAs and additional pro-neuronal factors, including NeuroD1 may cooperate with or replace the BAM factors during conversion of human fibroblasts into neurons. See, for example, Ambasudhan et al., 2011, Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions, Cell Stem Cell 9:113-118 ; Pang et al., 2011, Induction of human neuronal cells by defined transcription factors, Nature 476:220-223 ; also see Yoo et al., 2011, MicroRNA mediated conversion of human fibroblasts to neurons, Nature 476:228-231 .
  • Differentiated cells such as motor neurons may be dissociated and plated onto glass coverslips coated with poly-d-lysine and laminin.
  • Motor neurons may be fed with a suitable medium such as a neurobasal medium supplemented with N2, B27, GDNF, BDNF, and CTNF.
  • Cells may be maintained in a suitable medium such as an N2 medium (DMEM/F12 [1:1] supplemented with laminin [1 ⁇ g/mL; Invitrogen], FGF-2 [10 ng/ml; R&D Systems, Minneapolis, MN], and N2 supplement [1%; Invitrogen]), further supplemented with GDNF, BDNF, and CNTF, all at 10 ng/ml.
  • N2 medium DMEM/F12 [1:1] supplemented with laminin [1 ⁇ g/mL; Invitrogen], FGF-2 [10 ng/ml; R&D Systems, Minneapolis, MN]
  • Suitable media are described in Son et al., 2011, Conversion of mouse and human fibroblasts into functional spinal motor neurons, Cell Stem Cell 9:205-218 ; Vierbuchen et al., 2010, Direct conversion of fibroblasts to functional neurons by defined factors, Nature4 63:1035-1041 ; Kuo et al., 2003, Differentiation of monkey embryonic stem cells into neural lineages, Biology of Reproduction 68: 1727-1735 ; and Wernig et al., 2002, Tau EGFP embryonic stem cells: an efficient tool for neuronal lineage selection and transplantation. J Neuroscience Res 69:918-24 .
  • Methods of the disclosure may include causing the cell to express an optical reporter, observing a signature generated by the optical reporter, and comparing the observed signature to a control signature.
  • the control signature may be a disease free cell and can be obtained by obtaining a control cell that is also of the specific neural subtype and is genetically and phenotypically similar to the test cells.
  • a patient has a known mutation or allele at a certain locus-genetic editing is performed to generate a control cell line that but for the known mutation is isogenic with the test cell line.
  • genetic editing techniques can introduce a wild-type SHANK3 gene into the cell line to create a control cell line with a wild-type genotype and phenotype.
  • genome editing may be used to introduce a mutation of interest into a neuron in order to evaluate the phenotypic effect of the mutation and to investigate potential links to a condition such as Parkinson's disease.
  • Genetic or genome editing techniques may proceed via zinc-finger domain methods, transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeat (CRISPR) nucleases.
  • TALENs transcription activator-like effector nucleases
  • CRISPR clustered regularly interspaced short palindromic repeat
  • Genome editing techniques may be used to create test and control cells that are isogenic but-for a variant of interest.
  • genome editing techniques are applied to the iPS cells.
  • a second corrected line may be generated using zinc finger domains resulting in two otherwise isogenic lines.
  • diseased and corrected iPS cells may be differentiated into motor neurons using embryoid bodies according to the methods described above.
  • Genomic editing may be performed by any suitable method known in the art.
  • the chromosomal sequence encoding the target gene of interest may be edited using TALENs technology.
  • TALENS are artificial restriction enzymes generated by fusing a TAL effector DNA binding domain to a DNA cleavage domain.
  • genome editing is performed using CRISPR technology.
  • TALENs and CRISPR methods provide one-to-one relationship to the target sites, i.e. one unit of the tandem repeat in the TALE domain recognizes one nucleotide in the target site, and the crRNA or gRNA of CRISPR/Cas system hybridizes to the complementary sequence in the DNA target.
  • Methods can include using a pair of TALENs or a Cas9 protein with one gRNA to generate double-strand breaks in the target. The breaks are then repaired via non-homologous end-joining or homologous recombination (HR).
  • HR homologous recombination
  • TALENs uses a nonspecific DNA-cleaving nuclease fused to a DNA-binding domain that can be to target essentially any sequence.
  • target sites are identified and expression vectors are made.
  • the linearized expression vectors (e.g., by Notl) and used as template for mRNA synthesis.
  • a commercially available kit may be use such as the mMESSAGE mMACHINE SP6 transcription kit from Life Technologies (Carlsbad, CA). See Joung & Sander, 2013, TALENs: a wideliy applicable technology for targeted genome editing, Nat Rev Mol Cell Bio 14:49-55 .
  • CRISPR methodologies employ a nuclease, CRISPR-associated (Cas9), that complexes with small RNAs as guides (gRNAs) to cleave DNA in a sequence-specific manner upstream of the protospacer adjacent motif (PAM) in any genomic location.
  • CRISPR may use separate guide RNAs known as the crRNA and tracrRNA. These two separate RNAs have been combined into a single RNA to enable site-specific mammalian genome cutting through the design of a short guide RNA.
  • Cas9 and guide RNA (gRNA) may be synthesized by known methods.
  • Cas9/guide-RNA uses a non-specific DNA cleavage protein Cas9, and an RNA oligo to hybridize to target and recruit the Cas9/gRNA complex. See Chang et al., 2013, Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos, Cell Res 23:465-472 ; Hwang et al., 2013, Efficient genome editing in zebrafish using a CRISPR-Cas system, Nat.
  • genome editing is performed using zinc finger nuclease-mediated process as described, for example, in U.S. Pub. 2011/0023144 to Weinstein .
  • FIG. 3 gives an overview of a method 301 for zinc-finger nuclease mediated editing.
  • the method includes introducing into the iPS cell at least one RNA molecule encoding a targeted zinc finger nuclease 305 and, optionally, at least one accessory polynucleotide.
  • the cell includes target sequence 311.
  • the cell is incubated to allow expression of the zinc finger nuclease 305, wherein a double-stranded break 317 is introduced into the targeted chromosomal sequence 311 by the zinc finger nuclease 305.
  • a donor polynucleotide or exchange polynucleotide 321 is introduced.
  • Target DNA 311 along with exchange polynucleotide 321 may be repaired by an error-prone non-homologous end-joining DNA repair process or a homology-directed DNA repair process. This may be used to produce a control line with a control genome 315 that is isogenic to original genome 311 but for a changed site.
  • the genomic editing may be used to establish a control line (e.g., where the patient is known to have a certain mutation, the zinc finger process may revert the genomic DNA to wild type) or to introduce a mutation (e.g., non-sense, missense, or frameshift) or to affect transcription or expression.
  • a zinc finger nuclease comprises a DNA binding domain (i.e., zinc finger) and a cleavage domain (i.e., nuclease) and this gene may be introduced as mRNA (e.g., 5' capped, polyadenylated, or both).
  • Zinc finger binding domains may be engineered to recognize and bind to any nucleic acid sequence of choice. See, for example, Beerli & Barbas, 2002, Engineering polydactyl zinc-finger transcription factors, Nat. Biotechnol, 20:135-141 ; Pabo et al., 2001, Design and selection of novel Cys2His2 zinc finger proteins, Ann. Rev.
  • An engineered zinc finger binding domain may have a novel binding specificity compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection.
  • a zinc finger binding domain may be designed to recognize a target DNA sequence via zinc finger recognition regions (i.e., zinc fingers). See for example, U.S. Pat. Nos.
  • Exemplary methods of selecting a zinc finger recognition region may include phage display and two-hybrid systems, and are disclosed in U.S. Pat. 5,789,538 ; U.S. Pat. 5,925,523 ; U.S. Pat. 6,007,988 ; U.S. Pat. 6,013,453 ; U.S. Pat. 6,410,248 ; U.S. Pat. 6,140,466 ; U.S. Pat. 6,200,759 ; and U.S. Pat. 6,242,568 .
  • Zinc finger binding domains and methods for design and construction of fusion proteins are known to those of skill in the art and are described in detail in U.S. Pub. 2005/0064474 and U.S. Pub. 2006/0188987 .
  • Zinc finger recognition regions, multi-fingered zinc finger proteins, or combinations thereof may be linked together using suitable linker sequences, including for example, linkers of five or more amino acids in length. See, U.S. Pat. Nos. 6,479,626 ; 6,903,185 ; and 7,153,949 .
  • the zinc finger nuclease may use a nuclear localization sequence (NLS).
  • NLS nuclear localization sequence
  • a NLS is an amino acid sequence which facilitates targeting the zinc finger nuclease protein into the nucleus to introduce a double stranded break at the target sequence in the chromosome.
  • Nuclear localization signals are known in the art. See, for example, Makkerh, 1996, Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids, Current Biology 6:1025-1027 .
  • a zinc finger nuclease also includes a cleavage domain.
  • the cleavage domain portion of the zinc finger nucleases may be obtained from any suitable endonuclease or exonuclease such as restriction endonucleases and homing endonucleases. See, for example, Belfort & Roberts, 1997, Homing endonucleases: keeping the house in order, Nucleic Acids Res 25(17):3379-3388 .
  • a cleavage domain may be derived from an enzyme that requires dimerization for cleavage activity. Two zinc finger nucleases may be required for cleavage, as each nuclease comprises a monomer of the active enzyme dimer.
  • a single zinc finger nuclease may comprise both monomers to create an active enzyme dimer.
  • Restriction endonucleases present may be capable of sequence-specific binding and cleavage of DNA at or near the site of binding.
  • Certain restriction enzymes e.g., Type IIS
  • FokI active as a dimer, catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other.
  • the FokI enzyme used in a zinc finger nuclease may be considered a cleavage monomer.
  • two zinc finger nucleases, each comprising a FokI cleavage monomer may be used to reconstitute an active enzyme dimer. See Wah, et al., 1998, Structure of FokI has implications for DNA cleavage, PNAS 95:10564-10569 ; U.S. Pat. Nos. 5,356,802 ; 5,436,150 and 5,487,994 .
  • the cleavage domain may comprise one or more engineered cleavage monomers that minimize or prevent homo-dimerization, as described, for example, in U.S. Patent Publication Nos. 2005/0064474 , 2006/0188987 , and 2008/0131962 .
  • Genomic editing by the zinc finger nuclease-mediated process may include introducing at least one donor polynucleotide comprising a sequence into the cell.
  • a donor polynucleotide preferably includes the sequence to be introduced flanked by an upstream and downstream sequence that share sequence similarity with either side of the site of integration in the chromosome.
  • the upstream and downstream sequences in the donor polynucleotide are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide.
  • the donor polynucleotide will be DNA.
  • the donor polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, and may employ a delivery vehicle such as a liposome.
  • the sequence of the donor polynucleotide may include exons, introns, regulatory sequences, or combinations thereof.
  • the double stranded break is repaired via homologous recombination with the donor polynucleotide such that the desired sequence is integrated into the chromosome.
  • methods for genome editing include introducing into the cell an exchange polynucleotide (typically DNA) with a sequence that is substantially identical to the chromosomal sequence at the site of cleavage and which further comprises at least one specific nucleotide change.
  • an exchange polynucleotide typically DNA
  • a method such as TALENs, CRISPRs, or zinc fingers may be used to make a control cell line.
  • methods may be used to produce a cell line that is isogenic but for the SHANK3 mutation. While any such technology may be used, the following illustrates genome editing via zinc finger nucleases.
  • the sequence of the exchange polynucleotide will share enough sequence identity with the chromosomal sequence such that the two sequences may be exchanged by homologous recombination.
  • the sequence in the exchange polynucleotide comprises at least one specific nucleotide change with respect to the sequence of the corresponding chromosomal sequence. For example, one nucleotide in a specific codon may be changed to another nucleotide such that the codon codes for a different amino acid.
  • the sequence in the exchange polynucleotide may comprise one specific nucleotide change such that the encoded protein comprises one amino acid change.
  • a double stranded break introduced into the chromosomal sequence by the zinc finger nuclease is repaired, via homologous recombination with the exchange polynucleotide, such that the sequence in the exchange polynucleotide may be exchanged with a portion of the chromosomal sequence.
  • the presence of the double stranded break facilitates homologous recombination and repair of the break.
  • the exchange polynucleotide may be physically integrated or, alternatively, the exchange polynucleotide may be used as a template for repair of the break, resulting in the exchange of the sequence information in the exchange polynucleotide with the sequence information in that portion of the chromosomal sequence.
  • a portion of the endogenous chromosomal sequence may be converted to the sequence of the exchange polynucleotide.
  • At least one nucleic acid molecule encoding a zinc finger nuclease and, optionally, at least one exchange polynucleotide or at least one donor polynucleotide are delivered to the cell of interest.
  • Suitable methods of introducing the nucleic acids to the cell include microinjection, electroporation, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, heat shock transfection, lipofection, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions.
  • the method of inducing genomic editing with a zinc finger nuclease further comprises culturing the cell comprising the introduced nucleic acid to allow expression of the zinc finger nuclease.
  • Cells comprising the introduced nucleic acids may be cultured using standard procedures to allow expression of the zinc finger nuclease.
  • the cells are cultured at an appropriate temperature and in appropriate media with the necessary O2/CO2 ratio to allow the expression of the zinc finger nuclease.
  • Suitable non-limiting examples of media include M2, M16, KSOM, BMOC, and HTF media.
  • Standard cell culture techniques are described, for example, in Santiago et al, 2008, Targeted gene knockout in mammalian cells by using engineered zinc finger nucleases, PNAS 105:5809-5814 ; Moehle et al., 2007, Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases PNAS 104:3055-3060 ; Urnov et al., 2005, Highly efficient endogenous human gene correction using designed zinc-finger nucleases, Nature 435(7042):646-51 ; and Lombardo et al., 2007, Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery, Nat Biotechnol 25(11): 1298-306 .
  • the target sequence is edited.
  • the zinc finger nuclease recognizes, binds, and cleaves the target sequence in the chromosome.
  • the double-stranded break introduced by the zinc finger nuclease is repaired, via homologous recombination with the donor (or exchange) polynucleotide, such that the sequence in the donor polynucleotide is integrated into the chromosomal sequence (or a portion of the chromosomal sequence is converted to the sequence in the exchange polynucleotide).
  • a sequence may be integrated into the chromosomal sequence (or a portion of the chromosomal sequence may be modified).
  • an isogenic (but for the mutation of interest) control line can be generated.
  • a control cells are obtained from healthy individuals, i.e., without using genome editing on cells taken from the subject.
  • the control line can be used in the analytical methods described herein to generate a control signature for comparison to test data.
  • a control signature is stored on-file after having been previously generated and stored and the stored control signature is used (e.g., a digital file such as a graph or series of measurements stored in a non-transitory memory in a computer system).
  • a control signature could be generated by assaying a large population of subjects of known phenotype or genotype and storing an aggregate result as a control signature for later downstream comparisons.
  • Methods may be used to produce a cell line that is isogenic but for a mutation in a gene suspected of an association with Alzheimer's.
  • Genes suspected of an association with Alzheimer's include APOE; CLU (also known as APOJ); PICALM; E5-1; BDNF; ABCA7; MS4A6A/MS4A4E; EPHA1; CD33; CD2AP; SORL1; CR1; TREM2; APP; PS1; and PS2.
  • methods of the disclosure may be used to create a cell line with BDNF wild-type and a cell line with BDNF C270T (suspected Alzheimer's associated mutation per Kunugi et al., 2001, A novel polymorphism of the brain-derived neurotrophic factor (BDNF) gene associated with late-onset Alzheimer's disease, Mol Psych 6(1):83-86 ). While any such technology may be used, the following illustrates genome editing via zinc finger nucleases.
  • BDNF brain-derived neurotrophic factor
  • the patient's test cell line and the optional control line may be caused to express an optical reporter of neural or electrical activity.
  • neural activity include action potentials in a neuron or fusion of vesicles releasing neurotransmitters.
  • Exemplary electrical activity includes action potentials in a neuron, astrocyte or other electrically active cell.
  • Further examples of neural or electrical activity include ion pumping or release or changing ionic gradients across membranes.
  • Causing a cell to express an optical reporter of neural activity can be done with a fluorescent reporter of vesicle fusion.
  • Expressing an optical reporter of neural or electrical activity can include transformation with an optogenetic reporter.
  • the cell may be transformed with a vector comprising an optogenetic reporter and the cell may also be caused to express an optogenetic actuator by transformation.
  • the differentiated neurons are cultured (e.g., for about 4 days) and then infected with lentivirus bearing a genetically encoded optical reporter of neural activity and optionally an optical voltage actuator.
  • Any suitable optical reporter of neural activity may be used.
  • exemplary reporters include fluorescent reporters of transmembrane voltage differences, pHluorin-based reporters of synaptic vesicle fusion, and genetically encoded calcium indicators.
  • a genetically encoded voltage indicator is used.
  • Genetically encoded voltage indicators that may be used or modified for use with methods of the disclosure include FlaSh ( Siegel, 1997, A genetically encoded optical probe of membrane voltage.
  • a genetically encoded voltage indicator based on the paddle domain of a voltage-gated phosphatase is CiVSP ( Murata et al., 2005, Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor, Nature 435:1239-1243 ).
  • Another indicator is the hybrid hVOS indicator ( Chanda et al., 2005, A hybrid approach to measuring electrical activity in genetically specified neurons, Nat Neuroscience 8:1619-1626 ), which transduces the voltage dependent migration of dipicrylamine (DPA) through the membrane leaflet to "dark FRET" (fluorescence resonance energy transfer) with a membrane-targeted GFP.
  • DPA dipicrylamine
  • Optical reporters that may be suitable for use with the disclosure include those from the family of proteins of known microbial rhodopsins.
  • a reporter based on a microbial rhodopsin may provide high sensitivity and speed.
  • Suitable indicators include those that use the endogenous fluorescence of the microbial rhodopsin protein Archaerhodopsin 3 (Arch) from Halorubum sodomense. Arch resolves action potentials with high signal-to-noise (SNR) and low phototoxicity.
  • SNR signal-to-noise
  • a mutant form of Arch, D95N has been shown not to exhibit a hyperpolarizing current associated with some indicators.
  • Other mutant forms of Arch termed QuasAr!
  • Arch and the above-mentioned variants target eukaryotic membranes and can image single action potentials and subthreshold depolarization in cultured mammalian neurons. See Kralj et al, 2012, Optical recording of action potentials in mammalian neurons using a microbial rhodopsin, Nat Methods 9:90-95 . Thus Arch and variants of Arch such as Arch(D95N) may provide good optical reporters of neural activity according to aspects of the disclosure .
  • an improved variant of Arch such as QuasAr! or QuasAr2 is used.
  • QuasAr! comprises Arch with the mutations: P60S, T80S, D95H, D106H, and F161V.
  • QuasAr2 comprises Arch with the mutations: P60S, T80S, D95Q, D106H, and F161V.
  • Positions Asp95 and Asp 106 of Arch (which are structurally aligned with positions Asp85 and Asp96 of bacteriorhodopsin, and have been reported to play key roles in proton translocation during the photo cycle) are targets for modification because they flank the Schiff base in the proton-transport chain and are likely important in determining voltage sensitivity and speed.
  • the other mutations improve the brightness of the protein.
  • ER endoplasmic reticulum
  • TS
  • FIG. 4 presents a structural model of Quasar! based on homologous protein Arch-2 (PDB: 2EI4, described in Enami et al, 2006, Crystal structures of archaerhodopsin-1 and-2: Common structural motif in Archaeal light-driven proton pumps, J Mol Bio. 358:675-685 ). Mutations T80S and F161V are located in the periphery of the protein, while P60S is close to the Schiff base of the retinal chromophore. Given their location, T80S and F161V substitutions are unlikely to have a direct impact on the photo-physical properties of the protein, and are more likely to have a role in improving the folding efficiency.
  • QuasAr indicators may exhibit improved voltage sensitivity, response kinetics, membrane trafficking and diminished dependence of brightness on illumination intensity relative to Arch.
  • the fluorescence quantum yields of solubilized QuasAr1 and 2 may be 19- and 10-fold enhanced, respectively, relative to the non-pumping voltage indicator Arch(D95N).
  • QuasAr! may be 15-fold brighter than wild-type Arch, and QuasAr2 may be 3.3-fold brighter. Neither mutant shows the optical nonlinearity seen in the wild-type protein.
  • Fluorescence of Arch, QuasAr1, and QuasAr2 increase nearly linearly with membrane voltage between -100 mV and +50 mV. Fluorescence recordings may be acquired on an epifluorescence microscope, described in Kralj et al., 2012, Optical recording of action potentials in mammalian neurons using a microbial rhodopsin, Nat. Methods 9:90-95 .
  • QuasAr! and QuasAr2 each refer to a specific variant of Arch.
  • archaerhodopsin 3 (Arch) functions as a fast and sensitive voltage indicator.
  • Improved versions of Arch include the QuasArs ('quality superior to Arch'), described in Hochbaum et al., 2014.
  • QuasAr1 differs from wild-type Arch by the mutations P60S, T80S, D95H, D106H and F161V.
  • QuasAr2 differed from QuasAr! by the mutation H95Q.
  • QuasAr! and QuasAr2 report action potentials (APs).
  • FIG. 21 gives a comparison of AP waveforms as measured by the genetically encoded voltage indicator QuasAr2 and the voltage-sensitive dye, FluoVolt.
  • Cells are sparsely transfected with the QuasAr2 construct and then treated with FluoVolt dye.
  • QuasAr2 is excited by red laser light at a wavelength of 635 nm with fluorescence detection centered at 720 nm.
  • FluoVolt is excited by 488 nm laser light with fluorescence detection centered at 525 nm.
  • the top panel shows the simultaneously recorded AP waveforms from a cell expressing QuasAr2 (red line) and labeled with FluoVolt (green line).
  • the similarity of these traces establishes that QuasAr2 fluorescence accurately represents the underlying AP waveform.
  • the lower trace compares the FluoVolt AP waveform in the presence (FluoVolt+, QuasAr2+, green) and absence (FluoVolt+, QuasAr2-, cyan) of QuasAr2 expression.
  • the similarity of these two traces establishes that expression of QuasAr2 does not perturb the AP waveform.
  • FIG. 22 shows plots of the average waveforms from the traces in FIG. 21 .
  • FIG. 23 presents phototoxicity and photobleaching measurement of QuasAr2.
  • Cells are imaged under continuous red laser illumination ( ⁇ 50 W/cm2) for 500 s. Expanded views of the fluorescence recording are shown in the lower panels.
  • FIG. 24 graphs the average AP waveform shapes for the beginning (blue) and end (green) of the trace in FIG. 23 .
  • Arch and the above-mentioned variants target eukaryotic membranes and can image single action potentials and subthreshold depolarization in cultured mammalian neurons. See Kralj et al, 2012, Optical recording of action potentials in mammalian neurons using a microbial rhodopsin, Nat Methods 9:90-95 and Hochbaum et al., All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins, Nature Methods, 11, 825-833 (2014 ). Thus Arch and variants of Arch may provide good optical reporters of electrical activity according to aspects of the disclosure .
  • the disclosure provides optical reporters based on Archaerhodopsins that function in mammalian cells, including human stem cell-derived neurons. These proteins indicate electrical dynamics with sub-millisecond temporal resolution and sub-micron spatial resolution and may be used in non-contact, high-throughput, and high-content studies of electrical dynamics in cells and tissues using optical measurement of membrane potential. These reporters are broadly useful, particularly in eukaryotic, such as mammalian, including human cells.
  • the disclosure includes reporters based on Archaerhodopsin 3 (Arch 3) and its homologues.
  • Arch 3 is Archaerhodopsin from H. sodomense and it is known as a genetically-encoded reagent for high-performance yellow/green-light neural silencing.
  • Gene sequence at GenBank: GU045593.1 synthetic construct Arch 3 gene, complete cds. Submitted Sep. 28, 2009). These proteins localize to the plasma membrane in eukaryotic cells and show voltage-dependent fluorescence.
  • Fluorescence recordings may be acquired on an epifluorescence microscope, described in Hochbaum et al., All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins, Nature Methods, 11, 825-833 (2014 ).
  • Optical reporters of the disclosure show high sensitivity. In mammalian cells, Archaerhodopsin-based reporters show about 3-fold increase in fluorescence between -150 mV and +150 mV. The response is linear over most of this range. Membrane voltage can be measured with a precision of ⁇ 1 mV in a 1 s interval. Reporters of the disclosure show high speed. QuasAr1 shows 90% of its step response in 0.05 ms. The upstroke of a cardiac AP lasts approximately 1 ms, so the speeds of Arch-derived indicators meet the benchmark for imaging electrical activity. Reporters of the disclosure show high photo-stability and are comparable to GFP in the number of fluorescence photons produced prior to photobleaching.
  • the reporters may also show far red spectrum.
  • the Arch-derived voltage-indicating protein reporters sometimes referred to as genetically encoded voltage indicators (GEVIs)
  • GEVIs genetically encoded voltage indicators
  • the Arch-derived voltage-indicating protein reporters may be excited with a laser at wavelengths between 590 - 640 nm, and the emission is in the near infrared, peaked at 710 nm. The emission is farther to the red than any other existing fluorescent protein. These wavelengths coincide with low cellular auto-fluorescence. This feature makes these proteins particularly useful in optical measurements of action potentials as the spectrum facilitates imaging with high signal-to-noise ratio, as well as multi-spectral imaging in combination with other fluorescent probes.
  • Suitable optogenetic reporters include the two Arch variants dubbed Archer1 and Archer2 reported in Flytzanis, et al., 2014, Archaerohodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons, Nat Comm 5:4894 .
  • Archer1 and Archer2 exhibit enhanced radiance in response to 655 nm light have 3-5 times increased fluorescence and 55-99 times reduced photocurrents compared with Arch WT.
  • Archer1 (D95E and T99C) and Archer2 (D95E, T99C and A225M) may be used for voltage sensing.
  • Suitable optogenetic reporters include the Arch-derived voltage sensors with trafficking signals for enhanced localization as well as the Arch mutants dubbed Arch-EEN and Arch-EEQ reported in Gong et al., Enhanced Archaerhodopsin fluorescent protein voltage indicators, PLoSOne 8(6):e66959 .
  • Such reporters may include variants of Arch with the double mutations D95N-D106E (Arch-EEN) and D95Q-D106E (Arch-EEQ).
  • Suitable optogenetic reporters include sensors that use fluorescence resonance energy transfer (FRET) to combine rapid kinetics and the voltage dependence of the rhodopsin family voltage-sensing domains with the brightness of genetically engineered protein fluorophores.
  • FRET-opsin sensors offer good spike detection fidelity, fast kinetics, and high brightness.
  • FRET-opsin sensors are described in Gong et al., Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors, Nat Comm 5:3674 .
  • a suitable FRET-opsin may include a fusion of a bright fluorophore to act as a FRET donor to a Mac rhodopsin molecule to server as both the voltage sensing domain and the FRET acceptor.
  • Other sensors include the Accelerated Sensor of Action Potentials (ASAP1), a voltage sensor formed by insertion of a circularly permuted GFP into a chicken voltage-sensitive phosphatase. St-Pierre, 2014, High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor, Nat Neurosci 17(6):884 .
  • Other suitable reporters may include the ArcLight-derived probe dubbed Bongwoori and described in Piao et al., 2015, Combinatorial mutagenesis of the voltage-sensing domain enables the optical resolution of action potentials firing at 60 Hz by a genetically encoded fluorescent sensor of membrane potential, J Neurosci 35(1):372-385 .
  • the cells are transformed with an optical voltage actuator. This can occur, for example, simultaneously with transformation with the vector comprising the optogenetic reporter.
  • the far-red excitation spectrum of the QuasAr reporters suggests that they may be paired with a blue light-activated channelrhodopsin to achieve all-optical electrophysiology.
  • the channelrhodopsin should carry current densities sufficient to induce APs when only a subsection of a cell is excited.
  • light used for imaging the reporter should not activate the actuator, and light used for activating the actuator should not confound the fluorescence signal of the reporter.
  • an optical actuator and an optical reporter are spectrally orthogonal to avoid crosstalk and allow for simultaneous use.
  • Spectrally orthogonal systems are discussed in Carlson and Campbell, 2013, Circular permutated red fluorescent proteins and calcium ion indicators based on mCherry, Protein Eng Des Sel 26(12):763-772 .
  • a genetically-encoded optogenetic actuator is used.
  • One actuator is channelrhodopsin2 H134R, an optogenetic actuator described in Nagel, G. et al., 2005, Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses, Curr. Biol. 15, 2279-2284 .
  • SdChR Scherffelia dubia ChR
  • sdChR Scherffelia dubia Channelrhodopsin
  • sdChR selected from a screen of channelrhodopsins for its blue excitation peak (474 nm) and its large photocurrent relative to ChR2
  • sdChR The gene for Scherffelia dubia Channelrhodopsin (sdChR) (selected from a screen of channelrhodopsins for its blue excitation peak (474 nm) and its large photocurrent relative to ChR2) is synthesized with mouse codon optimization, a trafficking sequence from Kir2.1 is added to improve trafficking, and the mutation E154A is introduced.
  • CheRiff exhibits significantly decreased crosstalk from red illumination (to 10.5 ⁇ 2.8 pA) allowing its use in cells along with optogenetic reporters described herein. CheRiff shows good expression and membrane trafficking in cultured rat hippocampal neurons.
  • whole-cell illumination at only 22 ⁇ 10 mW/cm induces a photocurrent of 1 nA.
  • ChR2 H134R and to ChIEF under standard channelrhodopsin illumination conditions (488 nm, 500 mW/cm ).
  • Violet-activated channelrhodopsins can be simultaneously combined with yellow-excited Ca2+ indicators (e.g. jRCaMP1a, jRGECO1a, and R-CaMP2) and a red-excited voltage indicator, e.g. QuasAr2, for simultaneous monitoring of Ca2+ and voltage under optical stimulus conditions.
  • yellow-excited Ca2+ indicators e.g. jRCaMP1a, jRGECO1a, and R-CaMP2
  • a red-excited voltage indicator e.g. QuasAr2
  • a preferred violet-excited channelrhodopsin actuator is TsChR, derived from Tetraselmis striata (See Klapoetke et al., 2014, Independent optical excitation of distinct neural populations, Nat. Meth. 11, 338-346 (2014 )).
  • This channelrhodopsin actuator has a blue-shifted action spectrum with a peak at 435 nm.
  • PsChR derived from Platymonas subcordiformis (see Govorunova, Maria et al., 2013, Characterization of a highly efficient blue-shifted channelrhodopsin from the marine alga Platymonas subcordiformis, J Biol Chem 288(41):29911-29922 ).
  • PsChr has a blue-shifted action spectrum with a peak at 437 nm.
  • PsChR and TsChR are advantageously paired with red-shifted Ca2+ indicators and can be used in the same cell or same field of view as these red-shifted Ca2+ indicators without optical crosstalk.
  • the optogenetic reporters and actuators may be delivered in constructs described here as optopatch constructs delivered through the use of an expression vector.
  • Optopatch may be taken to refer to systems that perform functions traditionally associated with patch clamps, but via an optical input, readout, or both as provided for by, for example, an optical reporter or actuator.
  • An Optopatch construct may include a bicistronic vector for co-expression of CheRiff-eGFP and QuasAr1- or QuasAr2-mOrange2.
  • the QuasAr and CheRiff constructs may be delivered separately, or a bicistronic expression vector may be used to obtain a uniform ratio of actuator to reporter expression levels.
  • the genetically encoded reporter, actuator, or both may be delivered by any suitable expression vector using methods known in the art.
  • An expression vector is a specialized vector that contains the necessary regulatory regions needed for expression of a gene of interest in a host cell.
  • the gene of interest is operably linked to another sequence in the vector.
  • it is preferred that the viral vectors are replication defective, which can be achieved for example by removing all viral nucleic acids that encode for replication. A replication defective viral vector will still retain its infective properties and enters the cells in a similar manner as a replicating vector, however once admitted to the cell a replication defective viral vector does not reproduce or multiply.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector.
  • transcription control elements e.g. promoters, enhancers, and termination elements
  • viral vectors or virus-associated vectors are known in the art. Such vectors can be used as carriers of a nucleic acid construct into the cell. Constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral and lentiviral vectors, for infection or transduction into cells.
  • the vector may or may not be incorporated into the cell's genome.
  • the constructs may include viral sequences for transfection, if desired.
  • the construct may be incorporated into vectors capable of episomal replication, such as an Eptsein Barr virus (EPV or EBV) vector.
  • EPV Eptsein Barr virus
  • the inserted material of the vectors described herein may be operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence.
  • transcription of an inserted material is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene.
  • a recombinant cell containing an inducible promoter is used and exposed to a regulatory agent or stimulus by externally applying the agent or stimulus to the cell or organism by exposure to the appropriate environmental condition or the operative pathogen. Inducible promoters initiate transcription only in the presence of a regulatory agent or stimulus.
  • inducible promoters include the tetracycline response element and promoters derived from the beta-interferon gene, heat shock gene, metallothionein gene or any obtainable from steroid hormone-responsive genes.
  • inducible promoters which may be used in performing the methods of the present disclosure include those regulated by hormones and hormone analogs such as progesterone, ecdysone and glucocorticoids as well as promoters which are regulated by tetracycline, heat shock, heavy metal ions, interferon, and lactose operon activating compounds. See Gingrich and Roder, 1998, Inducible gene expression in the nervous system of transgenic mice, Annu Rev Neurosci 21:377-405 .
  • tissue specific expression has been well characterized in the field of gene expression and tissue specific and inducible promoters are well known in the art. These promoters are used to regulate the expression of the foreign gene after it has been introduced into the target cell.
  • a cell-type specific promoter or a tissue-specific promoter is used.
  • a cell-type specific promoter may include a leaky cell-type specific promoter, which regulates expression of a selected nucleic acid primarily in one cell type, but cause expression in other cells as well.
  • a neuron-specific enolase promoter can be used for expression of an exogenous gene specifically in neuronal cells.
  • the expression vector is a lentiviral vector.
  • Lentiviral vectors may include a eukaryotic promoter.
  • the promoter can be any inducible promoter, including synthetic promoters, that can function as a promoter in a eukaryotic cell.
  • the eukaryotic promoter can be, but is not limited to, CamKII ⁇ promoter, human Synapsin promoter, ecdysone inducible promoters, E1a inducible promoters, tetracycline inducible promoters etc., as are well known in the art.
  • the lentiviral vectors used herein can further comprise a selectable marker, which can comprise a promoter and a coding sequence for a selectable trait.
  • a selectable marker which can comprise a promoter and a coding sequence for a selectable trait.
  • Nucleotide sequences encoding selectable markers are well known in the art, and include those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of such sequences include, but are not limited to, those that encode thymidine kinase activity, or resistance to methotrexate, ampicillin, kanamycin, among others.
  • lentiviral vectors Use of lentiviral vectors is discussed in Wardill et al., 2013, A neuron-based screening platform for optimizing genetically-encoded calcium indicators, PLoS One 8(10):e77728 ; Dottori, et al., Neural development in human embryonic stem cells-applications of lentiviral vectors, J Cell Biochem 112(8):1955-62 ; and Diester et al., 2011, An optogenetic toolbox designed for primates, Nat Neurosci 14(3):387-97 . When expressed under a CaMKII ⁇ promoter in cultured rat hippocampal neurons the Optopatch construct exhibits high expression and good membrane trafficking of both CheRiff and QuasAr2.
  • the viral vector is an adeno-associated virus (AAV) vector.
  • AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell.
  • AAV adeno-associated virus
  • One suitable viral vector uses recombinant adeno-associated virus (rAAV), which is widely used for gene delivery in the CNS.
  • methods of the disclosure use a Cre-dependent expression system.
  • Cre-dependent expression includes Cre-Lox recombination, a site-specific recombinase technology that uses the enzyme Cre recombinase, which recombines a pair of short target sequences called the Lox sequences.
  • This system can be implemented without inserting any extra supporting proteins or sequences.
  • the Cre enzyme and the original Lox site called the LoxP sequence are derived from bacteriophage P1.
  • Bacteriophage P1 uses Cre-lox recombination to circularize and replicate its genomic DNA. This recombination strategy is employed in Cre-Lox technology for genome manipulation, which requires only the Cre recombinase and LoxP sites.
  • Methods may use a Cre recombinase-dependent viral vector for targeting tools such as channelrhodopsin-2 (ChR2) to specific neurons with expression levels sufficient to permit reliable photostimulation.
  • Optogenetic tools such as ChR2 tagged with a fluorescent protein such as mCherry (e.g., ChR2mCherry) or any other of the tools discussed herein are thus delivered to the cell or cells for use in characterizing those cells.
  • the delivery vector may include Cre and Lox.
  • the vector may further optionally include a Lox-stop-Lox (LSL) cassette to prevent expression of the transgene in the absence of Cre-mediated recombination.
  • LSL Lox-stop-Lox
  • the LoxP sites recombine, and a removable transcription termination Stop element is deleted. Removal of the stop element may be achieved through the use of AdenoCre, which allows control of the timing and location of expression.
  • LSL cassette is discussed in Jackson, et al., 2001, Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras, Genes & Dev 15:3243-3248 .
  • a construct of the disclosure uses a "flip-excision" switch, or FLEX switch (FLip EXicision), to achieve stable transgene inversion.
  • FLEX switch FLip EXicision
  • the FLEX switch is discussed in Schnutgen et al., 2003, A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse, Nat Biotechnol 21:562-565 .
  • the FLEX switch uses two pairs of heterotypic, antiparallel LoxP-type recombination sites which first undergo an inversion of the coding sequence followed by excision of two sites, leading to one of each orthogonal recombination site oppositely oriented and incapable of further recombination.
  • a FLEX switch provides high efficiency and irreversibility.
  • methods use a viral vector comprising rAAV-FLEX-rev-ChR2mCherry.
  • a vector may include FLEX and any other optogenetic tool discussed herein (e.g., rAAV-FLEX-QuasAr, rAAV-FLEX-CheRiff).
  • Cre-mediated inversion of the ChR2mCherry coding sequence results in the coding sequence being in the wrong orientation (i.e., rev-ChR2mCherry) for transcription until Cre inverts the sequence, turning on transcription of ChR2mCherry.
  • FLEX switch vectors are discussed in Atasoy et al., 2009, A FLEX switch targets channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping, J Neurosci 28(28):7025-7030 .
  • a viral vector such as Cre-Lox system with an optical reporter, optical actuator, or both (optionally with a FLEX switch and/or a Lox-Stop-Lox cassette) for labeling and stimulation of neurons allows for efficient photo-stimulation with only brief exposure (1 ms) to less than 100 ⁇ W focused laser light or to light from an optical fiber.
  • actuators, reporters, or other genetic material may be delivered using chemically-modified mRNA. It may be found and exploited that certain nucleotide modifications interfere with interactions between mRNA and toll-like receptor, retinoid-inducible gene, or both. Exposure to mRNAs coding for the desired product may lead to a desired level of expression of the product in the cells.
  • the neurons may be matured for 8-10 days post infection. Using microscopy and analytical methods described herein, the cell and its action potentials may be observed. For additional discussion, see U.S. Pub. 2013/0224756 and U.S. Pub. 2014/0295413 .
  • FIG. 25 presents schematic structures of optogenetic proteins used for stimulus and detection of voltage and intracellular Ca2+.
  • the diagrams show proteins homologous to CheRiff and QuasAr2. Stimulus of cells is achieved through 488 nm LED illumination of CheRiff.
  • the CheRiff construct is coupled to an eGFP tag for detection of CheRiff expression.
  • a fusion protein called CaViar consisting of QuasAr2 (Hochbaum et al., 2014) fused to GCaMP6f (Chen et al., 2013), is used for simultaneous voltage and Ca2+ imaging.
  • QuasAr2 is excited via red laser light.
  • GCaMP6f is excited via blue laser light.
  • Cells are separately transduced with either CheRiff or CaViar vectors.
  • FIG. 26 illustrates cellular plating configurations.
  • CheRiff cells solid cyan circles
  • CaViar cells solid red circles
  • the yellow dotted line indicates a microscope field of view.
  • cells are plated to spatially segregate CheRiff-expressing cells from CaViar-expressing cells to avoid optical crosstalk between the pulsed blue light used to periodically stimulate the CheRiff-expressing cells and the continuous blue light used to image the CaViar-expressing cells.
  • the CheRiff-expressing cells lay outside the imaging region.
  • Membrane potential is only one of several mechanisms of signaling within cells. One may correlate changes in membrane potential with changes in concentration of other species, such as Ca2+, H+ (i.e. pH), Na+, ATP, cAMP, NADH.
  • Ca2+, H+ i.e. pH
  • Na+ i.e. Na+
  • ATP cAMP
  • NADH a species of oxidized glutathione
  • the fusion of an Arch-based voltage indicator and a genetically encoded Ca2+ indicator is called CaViar (See Hou et al., 2014, Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents, Frontiers in physiology 5 ).
  • Concentration of ions such as sodium, potassium, chloride, and calcium can be simultaneously measured when the nucleic acid encoding the microbial rhodopsin is operably linked to or fused with an additional fluorescent analyte sensitive indicator; or when the microbial rhodopsin and the additional fluorescent analyte sensitive indicator are co-expressed in the same cell.
  • Red-shifted genetically encoded calcium indicators include R-GECO1 (See Zhao, Yongxin, et al. "An expanded palette of genetically encoded Ca2+ indicators.” Science 333.6051 (2011): 1888-1891 and Wu, Jiahui, et al.
  • Robotics and custom software may be used for screening large libraries or large numbers of conditions which are typically encountered in high throughput drug screening methods.
  • aspects of the disclosure provide for spatial separation of stimulating cells and reporter cells.
  • Expression of channelrhodopsin-based light-gated ion channels provides a means to achieve optical stimulus.
  • the blue light used to activate these channels may overlap spectrally with the light used to image most small-molecule and genetically encoded fluorescent reporters of physiological activity (e.g. gCaMP Ca2+ indicators, Percival ATP indicators, pHluorin pH indicators, VF2.1 .Cl voltage-sensitive dyes).
  • the light used to image these reporters may lead to off-target activation of all known channelrhodopsin actuators.
  • Methods of the disclosure allow a cellular culture to be optically stimulated while also using fluorescent reporters of any color, without optical crosstalk between the stimulus and the physiological measurement through the spatial separation of actuator cells and reporter cells.
  • One solution presented here comprises expressing channelrhodopsin actuators in one set of hiPSC-derived cells, and expressing reporters (e.g. CaViar dual-function Ca2+ and voltage reporter) in another set of cells. Flashes of blue light are delivered to the actuator cells, while continuous blue light is used to monitor the reporter cells. The actuator cells stimulate the reporter cells through synapses.
  • a key challenge is to identify and target the stimulus and the measurement light beams to the appropriate corresponding cells.
  • Methods of the disclosure provide at least two aspects of the solution to the problem of targeting separate stimulus and measurement light beams to the appropriate cells: a first approach based on spatial segregation and a second approach based on image processing and patterned illumination.
  • light is targeted to the actuator cells using spatial segregation of actuator and reporter-expressing cells.
  • Cells are independently infected with actuator and reporter and are re-plated in distinct but electrically contiguous regions.
  • Optical stimulus is delivered only to regions of the dish with cells expressing the actuator, and sensor measurements using any wavelength of light are recorded in regions of the dish away from cells expressing the actuator.
  • the actuator is CheRiff
  • the sensor is CaViar in human iPSC-derived neurons.
  • FIG. 27 shows cells expressing CheRiff plated in an annular region, 10 mm outer diameter, ⁇ 8 mm diameter.
  • the inner radius is set by a disk of polydimethyl siloxane (PDMS) adhered to the coverslip and the outer diameter is set by the edge of the chamber.
  • PDMS polydimethyl siloxane
  • the PDMS disk is then removed and cells expressing CaViar are plated throughout.
  • Stimulus is controlled by a blue LED whose illumination is confined to a small region of the actuating cells.
  • Voltage and calcium imaging are achieved with a red and blue laser, respectively, in a region free of CheRiff-expressing cells.
  • a second embodiment using patterned illumination light is targeted to the actuator cells using image processing and patterned illumination to separately target intermingled actuator- and reporter-expressing cells.
  • cells expressing either actuator or reporters are randomly intermixed.
  • cells are initially plated separately and caused to express either the actuator or the reporter. The cells are then lifted from their respective dishes, mixed, and co-plated onto the imaging dish.
  • cells are plated directly in the imaging chamber, and doubly infected with lentivirus encoding Cre-On actuator and a Cre-Off reporter. The cells are then infected sparsely with lentivirus encoding the Cre protein, so that in a sparse subset of cells the actuator is switched on and the reporter is switched off.
  • Cells expressing the actuator are identified via a recognizable marker, e.g. a fluorescent protein, or by their absence of fluorescence transients indicating presence of a reporter.
  • Optical stimulus is achieved by spatially patterning the excitation light using a digital micromirror device (DMD) to project flashes onto only those cells expressing the actuator.
  • DMD digital micromirror device
  • FIG. 5 diagrams an optical imaging apparatus 501 for patterned illumination.
  • a 488 nm blue laser beam is modulated in intensity by an acousto-optic modulator (not shown), and then reflected off a digital micromirror device (DMD) 505.
  • the DMD imparted a spatial pattern on the blue laser beam (used for CheRiff excitation) on its way into the microscope.
  • the micromirrors are re-imaged onto the sample 509, leading to an arbitrary user-defined spatio-temporal pattern of illumination at the sample. Simultaneous whole-field illumination with 640 nm red light excites fluorescence of the reporter.
  • the fluorescent protein serving as a recognizable marker of the cells expressing the actuator is imaged to determine a pattern of those actuator cells.
  • the digital coordinates of that image are used to control the DMD 505 so that the DMD 505 directs the blue 488 nm light only onto the actuator cells. Due to the precision of the patterned illumination provided by the DMD 505, the cells expressing the reporter are not exposed to the 488 nm light.
  • Cells expressing the reporter are imaged under continuous illumination, with the 640 nm light targeted via the DMD to illuminate only those cells expressing the reporter, and optionally continuous illumination at a wavelength of 488 nm to illuminate an additional reporter such as a GCaMP calcium indicator.
  • the actuator cells stimulate the reporter cells (e.g., across synapses).
  • the actuator cells comprise a first set of hiPSC-derived neurons expressing channelrhodopsin actuators and the reporter cells comprise a second set of hiPSC-derived neurons expressing reporters (e.g. QuasAr2 or CaViar dual-function Ca2+ and voltage reporter).
  • the foregoing (i) spatial segregation and (ii) patterned illumination methods provide for optical detection of changes in membrane potential, [Ca2+], or both, in optically stimulated neurons.
  • the described methods and techniques herein provide for the optical detection of the effects of compounds on cells such as cells with disease genotypes. Such detection allows for evaluating the effect of a compound or other stimulus on the phenotype of such cells.
  • MatTek dishes (MatTek corp.; 10mm glass diameter, #1.5) are coated with 10 ⁇ g/mL fibronectin (Sigma-Aldrich) in 0.1% gelatin overnight at 4 °C.
  • Trypsinized CaViar and CheRiff-expressing cells are first mixed at a ratio of 5:1 Ca Viar:CheRiff, and then pelleted.
  • the combined cells are re-suspended in 2.1 mL of maintenance medium and plated at a density of 2.5 ⁇ 104 cells/cm2 in 100 ⁇ L of plating medium to cover the entire glass surface. Cells are kept at 37° C in 5% CO2 overnight to adhere to the glass.
  • Maintenance medium (1.0 mL) is added to each dish and the cells are fed every 48 hours by removing 750 ⁇ L of medium from the dish and replacing with 750 ⁇ L fresh maintenance medium.
  • MatTek dishes (10 mm glass diameter) are prepared to segregate CheRiff-expressing cells from CaViar-expressing cells. This allows simultaneous calcium imaging and CheRiff stimulus, both with blue light, without optical crosstalk between the two functions.
  • 8 mm-diameter poly-dimethylsiloxane (PDMS) discs are treated with a solution of 10 ⁇ g/mL fibronectin in 0.1% gelatin on one side for 10 minutes at room temperature. The coated discs are then dried and then pressed onto the MatTek dish glass surface, slightly offset to one side. The remaining exposed area of the glass is then coated with 10 ⁇ g/mL fibronectin in 0.1% gelatin.
  • PDMS poly-dimethylsiloxane
  • Cells expressing the CheRiff are trypsinized according to the manufacturer's protocol and re-suspended in 50 ⁇ L of maintenance medium per dish. For plating, 50 ⁇ L of the CheRiff cells are then added to the exposed portion of the glass surface and allowed to sit for 40 minutes at 37 °C in 5% CO2 to allow the cells to adhere. The PDMS discs are then removed, the glass surface washed with 150 ⁇ L of maintenance media medium and the remaining volume aspirated. Trypsinized CaViar cells are then re-suspended in 100 ⁇ L of maintenance medium per dish and plated at a density of 2.0 ⁇ 104 cells/cm2 in 100 ⁇ L to cover the entire glass surface.
  • Methods of the disclosure may include stimulating the cells that are to be observed.
  • Stimulation may be direct or indirect (e.g., optical stimulation of an optical actuator or stimulating an upstream cell in synaptic communication with the cell(s) to be observed).
  • Stimulation may be optical, electrical, chemical, or by any other suitable method.
  • Stimulation may involve any pattern of a stimulation including, for example, regular, periodic pulses, single pulses, irregular patterns, or any suitable pattern.
  • Methods may include varying optical stimulation patterns in space or time to highlight particular aspects of cellular function. For example, a pulse pattern may have an increasing frequency.
  • imaging includes stimulating a neuron that expresses an optical actuator using pulses of light.
  • a neuron expressing an Optopatch construct may be exposed to whole-field illumination with pulses of blue light (10 ms, 25 mW/cm) to stimulate CheRiff, and simultaneous constant illumination with red light (800 W/cm) to excite fluorescence of QuasAr2.
  • the fluorescence of QuasAr2 may be imaged at a 1 kHz frame rate.
  • Key parameters include temporal precision with which single spikes can be elicited and recorded, signal-to-noise ratio (SNR) in fluorescence traces, and long-term stability of the reporter signal. Methods provided herein may be found to optimize those parameters.
  • measurements are made using a low-magnification microscope that images a 1.2 ⁇ 3.3 mm field of view with 3 ⁇ m spatial resolution and 2 ms temporal resolution.
  • measurements are made using a high-magnification microscope that images a 100 ⁇ m field of view with 0.8 ⁇ m spatial resolution and 1 ms temporal resolution.
  • a suitable instrument is an inverted fluorescence microscope, similar to the one described in the Supplementary Material to Kralj et al., 2012, Optical recording of action potentials in mammalian neurons using a microbial rhodopsin, Nat. Methods 9:90-95 .
  • illumination from a red laser 640 nm, 140 mW (Coherent Obis 637-140 LX) is expanded and focused onto the back-focal plane of a 60 ⁇ oil immersion objective, numerical aperture 1.45 (Olympus 1-U2B616).
  • FIG. 5 gives a functional diagram of components of an optical imaging apparatus 501 according to certain aspects.
  • a 488 nm blue laser beam is modulated in intensity by an acousto-optic modulator (not shown), and then reflected off a digital micromirror device (DMD) 505.
  • the DMD imparted a spatial pattern on the blue laser beam (used for CheRiff excitation) on its way into the microscope.
  • the micromirrors are re-imaged onto the sample 509, leading to an arbitrary user-defined spatiotemporal pattern of illumination at the sample. Simultaneous whole-field illumination with 640 nm red light excites fluorescence of the QuasAr reporter.
  • a blue laser 488 nm 50 mW (Omicron PhoxX) is sent through an acousto-optic modulator (AOM; Gooch and Housego 48058-2.5-.55-5W) for rapid control over the blue intensity.
  • AOM acousto-optic modulator
  • the beam is then expanded and modulated by DMD 505 with 608 ⁇ 684 pixels (Texas Instruments LightCrafter).
  • the DMD is controlled via custom software (Matlab) through a TCP/IP protocol.
  • the DMD chip is re-imaged through the objective onto the sample, with the blue and red beams merging via a dichroic mirror. Each pixel of the DMD corresponds to 0.65 ⁇ m in the sample plane.
  • a 532 nm laser is combined with the red and blue beams for imaging of mOrange2.
  • Software is written to map DMD coordinates to camera coordinates, enabling precise optical targeting of any point in the sample.
  • pixels on DMD 505 are mapped to pixels on the camera.
  • the DMD projects an array of dots of known dimensions onto the sample.
  • the camera acquires an image of the fluorescence.
  • Custom software locates the centers of the dots in the image, and creates an affine transformation to map DMD coordinates onto camera pixel coordinates.
  • a dual-band dichroic filter separates reporter (e.g., Arch) from excitation light.
  • a 531/40 nm bandpass filter (Semrock FF01-531/40-25) may be used for eGFP imaging; a 710/100 nm bandpass filter (Chroma, HHQ710/100) for Arch imaging; and a quad-band emission filter (Chroma ZET405/488/532/642m) for mOrange2 imaging and pre-measurement calibrations.
  • a variable-zoom camera lens (Sigma 18-200 mm f/3.5-6.3 II DC) is used to image the sample onto an EMCCD camera (Andor iXon + DU-860), with 128 ⁇ 128 pixels. Images may be first acquired at full resolution (128 ⁇ 128 pixels). Data is then acquired with 2 ⁇ 2 pixel binning to achieve a frame rate of 1,000 frames/s. For runs with infrequent stimulation (once every 5 s), the red illumination is only on from 1 s before stimulation to 50 ms after stimulation to minimize photobleaching. Cumulative red light exposure may be limited to ⁇ 5 min. per neuron.
  • Low magnification wide-field imaging is performed with a custom microscope system based around a 2 ⁇ , NA 0.5 objective (Olympus MVX-2). Illumination is provided by six lasers 640 nm, 500 mW (Dragon Lasers 635M500), combined in three groups of two. Illumination is coupled into the sample using a custom fused silica prism, without passing through the objective. Fluorescence is collected by the objective, passed through an emission filter, and imaged onto a scientific CMOS camera (Hamamatsu Orca Flash 4.0).
  • Blue illumination for channelrhodopsin stimulation is provided by a 473 nm, 1 W laser (Dragon Lasers), modulated in intensity by an AOM and spatially by a DMD (Digital Light Innovations DLi4130 - ALP HS).
  • the DMD is re-imaged onto the sample via the 2 ⁇ objective.
  • neurons may be imaged using wide-field illumination at 488 nm and eGFP fluorescence.
  • a user may select regions of interest on the image of the neuron, and specify a time course for the illumination in each region.
  • the software maps the user-selected pixels onto DMD coordinates and delivers the illumination instructions to the DMD.
  • the inverted fluorescence micro-imaging system records optically from numerous (e.g., 50) expressing cells or cell clusters in a single field of view.
  • the system may be used to characterize optically evoked firing patterns and AP waveforms in neurons expressing an Optopatch construct.
  • Each field of view is exposed to whole-field pulses of blue light to evoke activity (e.g., 0.5 s, repeated every 6 s, nine intensities increasing from 0 to 10 mW/cm).
  • Reporter fluorescence such as from QuasAr may be simultaneously monitored with whole-field excitation at 640 nm, 100 W/cm.
  • FIG. 6 illustrates a pulse sequence of red and blue light used to record action potentials under increasing optical stimulation.
  • neurons are imaged on a high resolution microscope with 640 nm laser (600 W/cm) for voltage imaging.
  • neurons are imaged on a high resolution microscope with 640 nm laser (600 W/cm) for voltage imaging and excited with a 488 nm laser (20-200 mW/cm).
  • Distinct firing patterns can be observed (e.g., fast adapting and slow-adapting spike trains).
  • System measurements can detect rare electrophysiological phenotypes that might be missed in a manual patch clamp measurement. Specifically, the cells' response to stimulation (e.g., optical actuation) may be observed. Instruments suitable for use or modification for use with methods of the disclosure are discussed in U.S. Pub. 2013/0170026 to Cohen .
  • populations of cells may be measured. For example, both diseased and corrected (e.g., by zinc finger domains) motor neurons may be measured.
  • a cell's characteristic signature such as a neural response as revealed by a spike train may be observed.
  • Fluorescence values are extracted from raw movies by any suitable method.
  • One method uses the maximum likelihood pixel weighting algorithm described in Kralj et al., 2012, Optical recording of action potentials in mammalian neurons using a microbial rhodopsin, Nat Methods 9:90-95 . Briefly, the fluorescence at each pixel is correlated with the whole-field average fluorescence. Pixels that showed stronger correlation to the mean are preferentially weighted. This algorithm automatically finds the pixels carrying the most information, and de-emphasizes background pixels.
  • a user defines a region comprising the cell body and adjacent neurites, and calculates fluorescence from the unweighted mean of pixel values within this region.
  • these two approaches give similar results.
  • direct averaging and the maximum likelihood pixel weighting approaches may be found to provide optimum signal-to-noise ratios.
  • An image or movie may contain multiple cells in any given field of view, frame, or image.
  • the segmentation is performed semi-automatically using an independent components analysis (ICA) based approach modified from that of Mukamel, et al., 2009, Automated analysis of cellular signals from large-scale calcium imaging data, Neuron 63:747-760 .
  • ICA independent components analysis
  • the ICA analysis can isolate the image signal of an individual cell from within an image.
  • FIG. 7-FIG. 10 illustrate the isolation of individual cells in a field of view. Individual cells are isolated in a field of view using an independent component analysis.
  • FIG. 7 shows an image that contains five neurons whose images overlap with each other.
  • the fluorescence signal at each pixel is an admixture of the signals from each of the neurons underlying that pixel.
  • the statistical technique of independent components analysis finds clusters of pixels whose intensity is correlated within a cluster, and maximally statistically independent between clusters. These clusters correspond to images of individual cells comprising the aggregate image of FIG. 7 .
  • the ICA time course has been broken into distinct contributions from each cell. Segmentation may reveal that the activities of the cells are strongly correlated, as expected for cells found together by ICA. In this case, the spike trains from the image segments are similar but show a progress over time as the cells signal one another.
  • FIG. 10 shows the individual filters used to map (and color code) individual cells from the original image.
  • action potentials can be identified as spike trains represented by the timing at which an interpolated action potential crosses a threshold at each pixel in the image. Identifying the spike train may be aided by first processing the data to remove noise, normalize signals, improve SNR, other pre-processing steps, or combinations thereof. Action potential signals may first be processed by removing photobleaching, subtracting a median filtered trace, and isolating data above a noise threshold. The spike train may then be identified using an algorithm based on sub-Nyquist action potential timing such as an algorithm based on the interpolation approach of Foust, et al., 2010, Action potentials initiate in the axon initial segment and propagate through axon collaterals reliably in cerebellar Purkinje neurons. J. Neurosci 30, 6891-6902 and Popovic et al, 2011, The spatio-temporal characteristics of action potential initiation in layer 5 pyramidal neurons: a voltage imaging study. J. Physiol. 589, 4167-4187 .
  • a sub-Nyquist action potential timing (SNAPT) algorithm highlights subcellular timing differences in AP initiation.
  • the algorithm may be applied for neurons expressing Optopatch1, containing a voltage reporter such as QuasAr!. Either the soma or a small dendritic region is stimulated. The timing and location of the ensuing APs is monitored.
  • FIG. 11 shows a patterned optical excitation being used to induce action potentials. Movies of individual action potentials are acquired (e.g., at 1,000 frames/ s), temporally registered, and averaged.
  • the first step in the temporal registration of spike movies is to determine the spike times. Determination of spike times is performed iteratively. A simple threshold-and-maximum procedure is applied to F(t) to determine approximate spike times, ⁇ T0 ⁇ . Waveforms in a brief window bracketing each spike are averaged together to produce a preliminary spike kernel K0(t). A cross-correlation of K0(t) with the original intensity trace F(t) is calculated. Whereas the timing of maxima in F(t) is subject to errors from single-frame noise, the peaks in the cross-correlation, located at times ⁇ T ⁇ , are a robust measure of spike timing. A movie showing the mean AP propagation may be constructed by averaging movies in brief windows bracketing spike times ⁇ T ⁇ .
  • the AP movie has high signal-to-noise ratio.
  • a reference movie of an action potential is thus created by averaging the temporally registered movies (e.g., hundreds of movies) of single APs. Each frame of the movie is then corrected by dividing by this baseline.
  • a spatial filter may include convolution with a Gaussian kernel, typically with a standard deviation of 1 pixel.
  • a temporal filter may be based upon Principal Components Analysis (PCA) of the set of single-pixel time traces. The time trace at each pixel is expressed in the basis of PCA eigenvectors. Typically the first 5 eigenvectors are sufficient to account for >99% of the pixel-to-pixel variability in AP waveforms, and thus the PCA Eigen-decomposition is truncated after 5 terms. The remaining eigenvectors represented uncorrelated shot noise.
  • PCA Principal Components Analysis
  • FIG. 12 shows eigenvectors resulting from a principal component analysis (PCA) smoothing operation performed to address noise. Photobleaching or other such non-specific background fluorescence may be addressed by these means.
  • PCA principal component analysis
  • FIG. 13 shows a relation between cumulative variance and eigenvector number.
  • FIG. 14 gives a comparison of action potential waveforms before and after the spatial and PCA smoothing operations.
  • a smoothly varying spline function may be interpolated between the discretely sampled fluorescence measurements at each pixel in this smoothed reference AP movie.
  • the timing at each pixel with which the interpolated AP crosses a user-selected threshold may be inferred with sub-exposure precision.
  • the user sets a threshold depolarization to track (represented as a fraction of the maximum fluorescence transient), and a sign for dV/dt (indicating rising or falling edge.
  • the filtered data is fit with a quadratic spline interpolation and the time of threshold crossing is calculated for each pixel.
  • FIG. 15 shows an action potential timing map.
  • the timing map may be converted into a high temporal resolution SNAPT movie by highlighting each pixel in a Gaussian time course centered on the local AP timing.
  • the SNAPT fits are converted into movies showing AP propagation as follows. Each pixel is kept dark except for a brief flash timed to coincide with the timing of the user-selected AP feature at that pixel. The flash followed a Gaussian time-course, with amplitude equal to the local AP amplitude, and duration equal to the cell-average time resolution, ⁇ . Frame times in the SNAPT movies are selected to be ⁇ 2-fold shorter than ⁇ . Converting the timing map into a SNAPT movie is for visualization; propagation information is in the timing map.
  • FIG. 16 shows the accuracy of timing extracted by the SNAPT algorithm for voltage at a soma via comparison to a simultaneous patch clamp recording.
  • FIG. 17 gives an image of eGFP fluorescence, indicating CheRiff distribution.
  • FIG. 18 presents frames from a SNAPT movie formed by mapping the timing information from FIG. 16 onto a high spatial resolution image from FIG. 17 .
  • the white arrows mark the zone of action potential initiation in the presumed axon initial segment (AIS).
  • FIGS. 16-18 demonstrate that methods of the disclosure can provide high resolution spatial and temporal signatures of cells expressing an optical reporter of neural activity.
  • cells may be fixed and stained for ankyrin-G, a marker of the AIS. Correlation of the SNAPT movies with the immunostaining images establish that the AP initiated at the distal end of the AIS.
  • the SNAPT technique does not rely on an assumed AP waveform; it is compatible with APs that change shape within or between cells.
  • the SNAPT movies show AP initiation from the soma in single neurites in measured cells.
  • the described methods are useful to reveal latencies between AP initiation at the AIS and arrival in the soma of 320 ⁇ 220 ⁇ s, where AP timing is measured at 50% maximum depolarization on the rising edge.
  • Optopatch can resolve functionally significant subcellular details of AP propagation. Discussion of signal processing may be found in Mattis et al., 2011, Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins, Nat. Meth. 9:159-172 ; and Mukamel et al., 2009, Automated analysis of cellular signals from large-scale calcium imaging data, Neuron 63(6):747-760 .
  • Methods of the disclosure are used to obtain a signature from the observed cell or cells tending to characterize a physiological parameter of the cell.
  • the measured signature can include any suitable electrophysiology parameter such as, for example, activity at baseline, activity under different stimulus strengths, tonic vs. phasic firing patterns, changes in AP waveform, others, or a combination thereof.
  • Measurements can include different modalities, stimulation protocols, or analysis protocols. Exemplarily modalities for measurement include voltage, calcium, ATP, or combinations thereof. Exemplary stimulation protocols can be employed to measure excitability, to measure synaptic transmission, to test the response to modulatory chemicals, others, and combinations thereof.
  • Methods of disclosure may employ various analysis protocols to measure: spike frequency under different stimulus types, action potential waveform, spiking patterns, resting potential, spike peak amplitude, others, or combinations thereof.
  • the imaging methods are applied to obtain a signature mean probability of spike for cells from the patient and may also be used to obtain a signature from a control line of cells such as a wild-type control (which may be produced by genome editing as described above so that the control and the wild-type are isogenic but for a single site).
  • the observed signature can be compared to a control signature and a difference between the observed signature and the expected signature corresponds to a positive diagnosis of the condition.
  • FIG. 19 shows a mean probability of spike of wild-type (WT) and mutant (SOD1) cells.
  • WT wild-type
  • SOD1 mutant
  • neurons and methods of the disclosure may be used to create disease models for in vitro investigation of neurological disorders such as epilepsy.
  • Neurons may be derived from iPSCs taken from individuals suffering from the neurological disorder or may be derived through genome editing by incorporating a genotype associated with the neurological disorder.
  • Disease models of the disclosure may be particularly useful in studying action potential generation and propagation and ion channel function before, during, and after an epileptic seizure.
  • a test mutation, suspected of being associated with a neurological disorder may be incorporated into a neuron through genome editing and the resulting modified neuron may be observed for signs of disease to evaluate the test mutation for links to the disease.
  • cell neuronal models of a disease may be chosen based on the exhibition of neuronal phenotypes associated with the disease, such as neurons with diminished voltage-gated sodium channel function compared to disease-free neurons or hyperexcitability. See Kearney, 2014, The More, the Better: Modeling Dravet Syndrome With Induced Pluripotent Stem Cell-Derived Neurons, Epilepsy Curr. 14(1): 33-34 .
  • Neuronal models of a disease such as epilepsy or Dravet syndrome may be selected based on genotypic characteristics such as a mutation to one or more of the following genes: SCN1A, WWOX, PRRT2, KCNC1 , STX1B, CARS2, STXB1 , KCNQ2, CDKL5, ARX, SPTAN, BRAT1 , KCNQ3, SCN2A, GABA receptors, NIPA2, CDKL5, PCDH19, and NAV1.1.
  • genotypic characteristics such as a mutation to one or more of the following genes: SCN1A, WWOX, PRRT2, KCNC1 , STX1B, CARS2, STXB1 , KCNQ2, CDKL5, ARX, SPTAN, BRAT1 , KCNQ3, SCN2A, GABA receptors, NIPA2, CDKL5, PCDH19, and NAV1.1.
  • neurons of the invention may be cultured for extended periods, such as 1 month, 2 months, 3 months, 4 months or longer in order to simulate aging. See Sánchez-Daocc, et al., 2012, Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease, EMBO Mol Med, 4: 380-395 .
  • Cells of the invention may be transformed with optical reporters of membrane potential, reporters of intracellular calcium levels, light-gated ion channels, or a combination thereof.
  • Cells may be monitored over time by inducing and observing action potentials and changes in intracellular calcium levels during disease progression in order to examine the neuronal effects of the subject condition, such as epilepsy.
  • Subject cells of the disease model may also be monitored pre and post application of various therapies in order to evaluate their effectiveness.
  • Test compounds can be evaluated as candidate therapies to determine suitability of a treatment prior to application to patient. E.g. one can test autism drugs to find the one that reverts the firing pattern back to wild-type, prevents or delays disease onset, or lessens disease symptoms.
  • the invention provides methods for identifying possible therapies for a patient by testing compounds, which systems and methods may be employed as personalized medicine. Due to the nature of the assays described herein, it may be possible to evaluate the effects of candidate therapeutic compounds on a per-patient basis thus providing a tool for truly personalized medicine. For example, an assay as described herein may reveal that a patient suffering from a certain disease has neurons or neural subtypes that exhibit a disease-type physiological phenotype under the assays described herein.
  • One or a number of different compounds may be applied to those neurons or neural subtypes. Cells that are exposed to one of those different compounds (or a combination of compounds) may exhibit a change in physiological phenotype from disease-type to normal. The compound or combination of compounds that affects the change in phenotype from disease-type to normal is thus identified as a candidate treatment compound for that patient.
  • FIG. 20 presents a system 1101 useful for performing methods of the invention.
  • Results from a lab e.g., transformed, converted patient cells
  • Imaging instrument 501 is operably coupled to an analysis system 1119, which may be a PC computer or other device that includes a processor 125 coupled to a memory 127.
  • a user may access system 1101 via PC 1135, which also includes a processor 125 coupled to a memory 127.
  • Analytical methods described herein may be performed by any one or more processor 125 such as may be in analysis system 1119, PC 1135, or server 1139, which may be provided as part of system 1101.
  • Server 1139 includes a processor 125 coupled to a memory 127 and may also include optional storage system 1143.
  • Any of the computing device of system 1101 may be communicably coupled to one another via network 1131.
  • Any, each, or all of analysis system 1119, PC 1135, and server 1139 will generally be a computer.
  • a computer will generally include a processor 125 coupled to a memory 127 and at least one input/output device.
  • a processor 125 will generally be a silicon chip microprocessor such as one of the ones sold by Intel or AMD.
  • Memory 127 may refer to any tangible, non-transitory memory or computer readable medium capable of storing data or instructions, which-when executed by a processer 125-cause components of system 1101 to perform methods described herein.
  • Typical input/output devices may include one or more of a monitor, keyboard, mouse, pointing device, network card, Wi-Fi card, cellular modem, modem, disk drive, USB port, others, and combinations thereof.
  • network 1131 will include hardware such as switches, routers, hubs, cell towers, satellites, landlines, and other hardware such as makes up the Internet.
  • Example 1 Optical differentiation of a motor neuron model of amyotrophic lateral sclerosis (ALS) arising from a monogenic mutation in the SOD1 gene (SOD1A4V).
  • ALS amyotrophic lateral sclerosis
  • ALS is a fatal neurodegenerative disease that affects pyramidal neurons in the motor cortex and lower motor neurons that originate in the brainstem and spinal cord. See Musaro, 2010, State of the art and the dark side of amyotrophic lateral sclerosis, WJBC 1(5):62-68 . Typical manifestations include degeneration of motor neurons leading to muscle weakness and atrophy, speech and swallowing disabilities, paralysis, and death by respiratory failure. ALS is classified into sporadic or familial forms. It is thought that many of the familiar forms are caused by mutations in the Cu/Zn superoxide dismutase-1 (SOD1) protein.
  • SOD1 Cu/Zn superoxide dismutase-1
  • C9orf72 Another gene that may be used is C9orf72 where an incompletely penetrant mutation is sometimes associated with symptoms.
  • SOD1 converts the toxic mitochondrial by-product superoxide into water or hydrogen peroxide.
  • Evidence suggests SOD1 mutations are gain-of-function mutations.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Neurology (AREA)
  • Toxicology (AREA)
  • Neurosurgery (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Physiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Claims (7)

  1. Verfahren für ein Screening einer Verbindung für eine Epilepsiebehandlung, das Verfahren umfassend:
    Präsentieren einer Verbindung zu einer Probe, umfassend ein Neuron, das ein oder mehrere genotypische oder phänotypische Eigenschaften von Epilepsie aufweist, wobei die genotypische Eigenschaft eine Mutation in einem Gen umfasst, das aus der Gruppe ausgewählt ist, bestehend aus SCN1A, WWOX, PRRT2, KCNC1, STX1B, CARS2, STXB1, KCNQ2, CDKL5, ARX, SPTAN, BRAT1, KCNQ3, SCN2A, GABA-Rezeptoren, NIPA2, CDKL5, PCDH19 und NAV1. 1 oder wobei die phänotypische Eigenschaft aus der Gruppe ausgewählt ist, bestehend aus einer verminderten spannungsgesteuerten Natriumkanalfunktion im Vergleich zu krankheitsfreien Neuronen und Hyperanregungsfähigkeit, wobei das Neuron einen optischen Reporter eines elektrischen Membranpotentials, einen lichtgesteuerten lonenkanal, der ein Algenkanalrhodopsin umfasst, und ein Protein, das eine Änderung eines intrazellulären Kalziumspiegels meldet, der eine GCaMP-Variante umfasst, exprimiert, und wobei das Neuron durch ein zweites Neuron stimuliert wird, das den lichtgesteuerten lonenkanal exprimiert und wobei das zweite Neuron ebenso den optischen Reporter des elektrischen Membranpotentials exprimiert;
    Empfangen, über ein Mikroskopiesystem, eines durch den optischen Reporter erzeugten optischen Signals als Reaktion auf optische Stimulationen der Probe nach der Präsentation der Verbindung, wobei die Lichtimpulse, die eine optische Stimulation abgeben, keine Fluoreszenz der Reporter induzieren; wobei das Licht, das verwendet wird, um die Reporter abzubilden, den lichtgesteuerten lonenkanal nicht auslöst; und die Fluoreszenz jedes Reporters unterscheidbar ist, wodurch ein Übersprechen vermieden wird; Erfassen einer Änderung der Aktionspotenzialwellenform (AP-Wellenform) und einer Änderung des intrazellulären Kalziumspiegels bei einer Exposition des Neurons zu der Verbindung; und
    Identifizieren der Verbindung als einen Kandidaten für eine Epilepsiebehandlung basierend auf dem optischen Signal.
  2. Verfahren nach Anspruch 1, wobei das Mikroskopiesystem eine digitale Mikrospiegelvorrichtung umfasst, die optische Stimulation bereitstellt.
  3. Verfahren nach Anspruch 1, wobei das Mikroskopiesystem ferner eine ladungsgekoppelte Vorrichtungskamera umfasst, die konfiguriert ist, um das optische Signal von dem Neuron zu erfassen.
  4. Verfahren nach Anspruch 1, ferner umfassend ein räumliches Strukturieren einer Vielzahl von Neuronen in einer Zellkultur auf einem Substrat.
  5. Verfahren nach Anspruch 1, wobei die Verbindung Lacosamid oder Levetiracetam umfasst.
  6. Verfahren nach Anspruch 1, wobei der Identifizierungsschritt ein Vergleichen des optischen Signals der Probe mit einem optischen Signal umfasst, das von einer Kontrollzelle erhalten wird.
  7. Verfahren nach Anspruch 1, wobei das Übersprechen durch ein Auswählen eines Auslösers und von Reportern mit wenig spektraler Überlappung vermieden wird; vorzugsweise wobei der Auslöser durch violettes Licht aktiviert wird, der Kalzium-Reporter durch gelbes Licht angeregt wird und orangenes Licht emittiert, und der Spannungs-Reporter durch rotes Licht angeregt wird und Nahinfrarotlicht emittiert.
EP15720856.2A 2014-04-22 2015-04-21 Optogenetische analyse von verbindungen Active EP3134728B1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201461982589P 2014-04-22 2014-04-22
PCT/US2015/026858 WO2015164363A1 (en) 2014-04-22 2015-04-21 Optogenetic analysis of compounds

Publications (2)

Publication Number Publication Date
EP3134728A1 EP3134728A1 (de) 2017-03-01
EP3134728B1 true EP3134728B1 (de) 2023-03-29

Family

ID=53053109

Family Applications (3)

Application Number Title Priority Date Filing Date
EP15725136.4A Withdrawn EP3134729A1 (de) 2014-04-22 2015-04-21 Analyse von verbindungen für schmerz- und sensorische erkrankungen
EP15720856.2A Active EP3134728B1 (de) 2014-04-22 2015-04-21 Optogenetische analyse von verbindungen
EP15720177.3A Withdrawn EP3134727A1 (de) 2014-04-22 2015-04-21 Diagnostische verfahren für neuronale erkrankungen

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP15725136.4A Withdrawn EP3134729A1 (de) 2014-04-22 2015-04-21 Analyse von verbindungen für schmerz- und sensorische erkrankungen

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP15720177.3A Withdrawn EP3134727A1 (de) 2014-04-22 2015-04-21 Diagnostische verfahren für neuronale erkrankungen

Country Status (5)

Country Link
US (7) US20150301028A1 (de)
EP (3) EP3134729A1 (de)
JP (4) JP2017518738A (de)
CA (3) CA2946385A1 (de)
WO (4) WO2015164363A1 (de)

Families Citing this family (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10860683B2 (en) 2012-10-25 2020-12-08 The Research Foundation For The State University Of New York Pattern change discovery between high dimensional data sets
WO2015164363A1 (en) 2014-04-22 2015-10-29 Q-State Biosciences, Inc. Optogenetic analysis of compounds
WO2016187543A1 (en) 2015-05-21 2016-11-24 Q-State Biosciences, Inc. Optogenetics microscope
US10568516B2 (en) * 2015-06-22 2020-02-25 The Board Of Trustees Of The Leland Stanford Junior University Methods and devices for imaging and/or optogenetic control of light-responsive neurons
US11248212B2 (en) 2015-06-30 2022-02-15 StemoniX Inc. Surface energy directed cell self assembly
JP2018537073A (ja) 2015-10-15 2018-12-20 ステモニックス インコーポレイティド 中空繊維バイオリアクターを用いた細胞の製造方法
WO2020206362A1 (en) 2019-04-04 2020-10-08 Inscopix, Inc. Multi-modal microscopic imaging
KR101906577B1 (ko) * 2016-08-23 2018-10-10 경북대학교 산학협력단 척수 내 성상세포의 광유전학적 자극을 통한 통증 유발 방법 및 이를 이용한 통증 예방 또는 치료제의 스크리닝 방법
WO2018052788A1 (en) * 2016-09-13 2018-03-22 Inscopix, Inc. Adapter for microscopic imaging
CN108018314A (zh) * 2016-11-03 2018-05-11 优美佳生物技术有限公司 用于细胞重编程的方法和组合物
US11385236B2 (en) 2016-11-30 2022-07-12 Howard Hughes Medical Institute Chemigenetic voltage indicators
US20210138039A1 (en) 2017-03-09 2021-05-13 Q-State Biosciences, Inc. Pain response mediator compositions and methods for making and using same
EP3612088A4 (de) * 2017-04-21 2021-03-31 AnaBios Corporation Verfahren zur identifizierung von subtypen sensorischer neuronen in ex-vivo-zubereitungen
US11227692B2 (en) * 2017-12-28 2022-01-18 International Business Machines Corporation Neuron model simulation
US10829727B2 (en) 2018-04-11 2020-11-10 Trustees Of Boston University Engineered platform to generate 3D cardiac tissues
US10981578B2 (en) * 2018-08-02 2021-04-20 GM Global Technology Operations LLC System and method for hardware verification in an automotive vehicle
JP6567153B1 (ja) * 2018-09-28 2019-08-28 学校法人東北工業大学 対象化合物の特性の予測を行うための方法、コンピュータシステム、プログラム
US10630917B1 (en) * 2018-09-28 2020-04-21 Massachusetts Institute Of Technology Apparatus and methods for all-CMOS compressive sensing
CN109813912B (zh) * 2019-01-04 2021-12-28 深圳大学 一组血清差异蛋白组合在制备用于检测孤独症的试剂中的应用
US20220401391A1 (en) * 2019-05-09 2022-12-22 Apkarian Tech Llc Methods and compositions for treating pain
WO2021050593A1 (en) * 2019-09-09 2021-03-18 Scribe Therapeutics Inc. Compositions and methods for the targeting of sod1
DE102020110192A1 (de) * 2020-04-14 2021-10-14 UMS - Umwelt-, Membran- und Sensortechnik GmbH & Co. KG Verfahren und Vorrichtung zur störquellenunabhängigen lumineszenzbasierten Messung eines Analyten
EP4189065A1 (de) * 2020-07-30 2023-06-07 Allen Institute Systeme, vorrichtungen und verfahren zum screening von verbindungen mittels prädiktiver markierung
WO2022198072A1 (en) * 2021-03-18 2022-09-22 Brown University Characterizing the binding interactions between musk and bmp receptors
US20220357313A1 (en) * 2021-05-04 2022-11-10 Q-State Biosciences, Inc. Drug fingerprinting
WO2022266346A1 (en) * 2021-06-17 2022-12-22 Q-State Biosciences, Inc. Methods for producing neural cells
CA3224332A1 (en) * 2021-06-17 2022-12-22 Q-State Biosciences, Inc. Therapeutic oligonucleotide methods

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140295413A1 (en) * 2010-08-23 2014-10-02 President And Fellows Of Harvard College Systems, methods, and workflows for optogenetics analysis

Family Cites Families (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3975084A (en) 1973-09-27 1976-08-17 Block Engineering, Inc. Particle detecting system
US5487994A (en) 1992-04-03 1996-01-30 The Johns Hopkins University Insertion and deletion mutants of FokI restriction endonuclease
US5356802A (en) 1992-04-03 1994-10-18 The Johns Hopkins University Functional domains in flavobacterium okeanokoites (FokI) restriction endonuclease
US5436150A (en) 1992-04-03 1995-07-25 The Johns Hopkins University Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease
US6242568B1 (en) 1994-01-18 2001-06-05 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6140466A (en) 1994-01-18 2000-10-31 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
EP0781331B1 (de) 1994-08-20 2008-09-03 Gendaq Limited Verbesserung in bezug auf bindungsproteine bei der erkennung von dna
US5789538A (en) 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
US5591444A (en) 1995-07-28 1997-01-07 Isolagen Technologies, Inc. Use of autologous dermal fibroblasts for the repair of skin and soft tissue defects
US5925523A (en) 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof
DE19644662C2 (de) 1996-10-25 2000-04-13 Leica Microsystems Beleuchtungseinrichtung für ein Mikroskop
US6410248B1 (en) 1998-01-30 2002-06-25 Massachusetts Institute Of Technology General strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites
AU746454B2 (en) 1998-03-02 2002-05-02 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6453242B1 (en) 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
US7019376B2 (en) 2000-08-11 2006-03-28 Reflectivity, Inc Micromirror array device with a small pitch size
AU2002211142A1 (en) * 2000-10-26 2002-05-06 Actar Ab Screening method using a g coupled receptor associated with a specific g protein
JP5048899B2 (ja) 2001-09-28 2012-10-17 オリンパス株式会社 顕微鏡
US6885492B2 (en) 2001-11-08 2005-04-26 Imaginative Optics, Inc. Spatial light modulator apparatus
FI112540B (fi) 2002-02-13 2003-12-15 Janesko Oy Menetelmä väliaineessa olevien partikkeleiden valaisemiseksi optista analysointia varten ja optinen partikkelianalysaattori
DE10216005A1 (de) 2002-04-11 2003-10-30 Max Planck Gesellschaft Verwendung von biologischen Photorezeptoren als direkt lichtgesteuerte Ionenkanäle
DE10362002B4 (de) 2003-06-23 2006-10-12 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Adulte pluripotente Stammzellen
US7888121B2 (en) 2003-08-08 2011-02-15 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US8409861B2 (en) 2003-08-08 2013-04-02 Sangamo Biosciences, Inc. Targeted deletion of cellular DNA sequences
US20070087959A1 (en) 2003-08-19 2007-04-19 University Of Pittsburgh Of The Commonwealth System Of Higher Education Method of inducing biomineralization method of inducing bone regeneration and methods related thereof
FR2890446B1 (fr) 2005-09-05 2008-04-18 Cis Bio Internat Sa Methode de detection d'interaction intracellulaire entre bio-molecules
JP2009513159A (ja) * 2005-10-31 2009-04-02 ジヤンセン・フアーマシユーチカ・ナームローゼ・フエンノートシヤツプ Ｔｒｐｖ２のモジュレーターを同定するための組成物および方法
US20070208040A1 (en) * 2006-03-02 2007-09-06 Elfatih Elzein A2a adenosine receptor antagonists
EP2213731B1 (de) 2006-05-25 2013-12-04 Sangamo BioSciences, Inc. Varianten von Foki Spaltungs-Halbdomänen
US7838302B2 (en) 2006-08-07 2010-11-23 President And Fellows Of Harvard College Sub-diffraction limit image resolution and other imaging techniques
ATE455312T1 (de) 2007-02-05 2010-01-15 Olympus Corp Laser-scanning-mikroskop
US8257969B2 (en) 2007-04-12 2012-09-04 The Provost Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Genetic suppression and replacement
EP2025615A1 (de) 2007-08-03 2009-02-18 Nestec S.A. Behälter mit Mitteln zur Abgabe eines akustischen Signals beim Erreichen einer korrekten Verschlussposition
EP2028268A1 (de) 2007-08-20 2009-02-25 Université Libre De Bruxelles Erzeugung von Nervenzellen aus pluripotenten Stammzellen
CL2007002908A1 (es) 2007-10-09 2008-05-30 Univ Concepcion Procedimiento electroquimico in vitro para evaluar capacidad neurotoxica de estructuras, principios activos y/o compuestos de interes en celulas vivas y que comprende medir y cuantificar los parametros electricos en membranas expuestas a dichos agreg
JP5436434B2 (ja) 2007-10-11 2014-03-05 バーテックス ファーマシューティカルズ インコーポレイテッド 電位開口型ナトリウムチャネルの阻害剤として有用なアミド
US8562658B2 (en) 2007-12-06 2013-10-22 Technion Research & Development Foundation Limited Method and system for optical stimulation of neurons
FR2927633B1 (fr) 2008-02-19 2012-07-13 Commissariat Energie Atomique Systeme et procede de culture clonale de cellules epitheliales et leurs applications.
EP3165534B1 (de) 2008-04-23 2018-09-26 The Board of Trustees of The Leland Stanford Junior University Systeme, verfahren und zusammensetzungen zur optischen reizung von zielzellen
EP2334315B1 (de) 2008-09-03 2023-01-18 NoNO Inc. Mittel und verfahren zur schmerzbehandlung
US20120053084A1 (en) * 2008-09-04 2012-03-01 Galenea Corp. Synaptic vesicle cycling assays and systems
NZ602416A (en) 2008-11-14 2014-08-29 Univ Leland Stanford Junior Optically-based stimulation of target cells and modifications thereto
US20110023144A1 (en) 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in amyotrophyic lateral sclerosis disease
JP5340799B2 (ja) 2009-05-08 2013-11-13 オリンパス株式会社 レーザ走査型顕微鏡
DE102009043747A1 (de) 2009-09-30 2011-03-31 Carl Zeiss Microlmaging Gmbh Verfahren zur Erzeugung eines Mikroskopbildes und Mikroskop
WO2011050470A1 (en) 2009-10-29 2011-05-05 Mcmaster University Generating induced pluripotent stem cells and progenitor cells from fibroblasts
WO2011056975A2 (en) 2009-11-06 2011-05-12 Howard Hughes Medical Institute Genetically encoded calcium indicators and methods of use
US20130170026A1 (en) 2010-01-15 2013-07-04 Adam E. Cohen Convex Lens-Induced Confinement for Measuring Distributions of Molecular Size
KR101198476B1 (ko) 2010-08-31 2012-11-06 연세대학교 산학협력단 나노 구조 기반의 초고해상도 영상 방법 및 장치
US8532398B2 (en) 2010-03-26 2013-09-10 General Electric Company Methods and apparatus for optical segmentation of biological samples
EP2609425A1 (de) * 2010-08-23 2013-07-03 President and Fellows of Harvard College Optogenetische sonden zur messung des membranpotenzials
WO2012054484A1 (en) 2010-10-18 2012-04-26 The Research Foundation Of State University Of New York Optical control of cardiac function
WO2012112737A2 (en) 2011-02-16 2012-08-23 The Regents Of The University Of California Alzheimer's disease cellular model for diagnostic and therapeutic development
US8580937B2 (en) 2011-03-14 2013-11-12 Board Of Regents Of The University Of Texas System cDNA-derived nucleic acids encoding red-shifted channelrhodopsins
CA2856423A1 (en) 2011-11-23 2013-05-30 President And Fellows Of Harvard College Systems and methods for imaging at high spatial and/or temporal precision
EP2720075B1 (de) 2012-10-12 2017-11-29 Spectral Applied Research Inc. Interne Totalreflexionsfluoreszenzmikroskopie (TIRF) über mehrere Wellenlängen gleichzeitig
US8697359B1 (en) * 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
WO2015164363A1 (en) 2014-04-22 2015-10-29 Q-State Biosciences, Inc. Optogenetic analysis of compounds

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140295413A1 (en) * 2010-08-23 2014-10-02 President And Fellows Of Harvard College Systems, methods, and workflows for optogenetics analysis

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
EPI4K CONSORTIUM & EPILEPSY PHENOME/GENOME PROJECT: "De novo mutations in epileptic encephalopathies", NATURE, vol. 501, no. 7466, 11 August 2013 (2013-08-11), pages 217 - 221, XP055429368, ISSN: 0028-0836, DOI: 10.1038/nature12439 *
FRANCESCO NICITA ET AL: "The genetics of monogenic idiopathic epilepsies and epileptic encephalopathies", SEIZURE, BAILLIERE TINDALL, LONDON, GB, vol. 21, no. 1, 9 August 2011 (2011-08-09), pages 3 - 11, XP028344150, ISSN: 1059-1311, [retrieved on 20110817], DOI: 10.1016/J.SEIZURE.2011.08.007 *
FRANCESCO NICITA ET AL: "The genetics of monogenic idiopathic epilepsies and epileptic encephalopathies", SEIZURE, BAILLIERE TINDALL, LONDON, GB, vol. 21, no. 1, 9 August 2011 (2011-08-09), pages 3 - 11, XP028344150, ISSN: 1059-1311, Retrieved from the Internet <URL:https://reader.elsevier.com/reader/sd/pii/S1059131111002123?token=DAE062C17D2E20C092A82640B77AA8009749BDF2C54ADA8E866A8A6EC566609EA937EA15BFCA0DC34A9C8ABE1981698F&originRegion=eu-west-1&originCreation=20211222103002> [retrieved on 20110817], DOI: 10.1016/J.SEIZURE.2011.08.007 *
JACK M PARENT ET AL: "Reprogramming patient-derived cells to study the epilepsies", NATURE NEUROSCIENCE, vol. 18, no. 3, 24 February 2015 (2015-02-24), US, pages 360 - 366, XP055428863, ISSN: 1097-6256, DOI: 10.1038/nn.3944 *
JIAO JIAO ET AL: "Modeling Dravet syndrome using induced pluripotent stem cells (iPSCs) and directly converted neurons", HUMAN MOLECULAR GENETICS, vol. 22, no. 21, 16 June 2013 (2013-06-16), gb, pages 4241 - 4252, XP055428868, ISSN: 0964-6906, DOI: 10.1093/hmg/ddt275 *
NORIMICHI HIGURASHI ET AL: "A human Dravet syndrome model from patient induced pluripotent stem cells", MOLECULAR BRAIN, BIOMED CENTRAL LTD, LONDON UK, vol. 6, no. 1, 2 May 2013 (2013-05-02), pages 19, XP021151579, ISSN: 1756-6606, DOI: 10.1186/1756-6606-6-19 *
RHYS H. THOMAS ET AL: "The hidden genetics of epilepsy-a clinically important new paradigm", NATURE REVIEWS. NEUROLOGY, vol. 10, no. 5, 15 April 2014 (2014-04-15), US, pages 283 - 292, XP055429364, ISSN: 1759-4758, DOI: 10.1038/nrneurol.2014.62 *

Also Published As

Publication number Publication date
US20190025291A1 (en) 2019-01-24
WO2015164383A1 (en) 2015-10-29
CA2946378A1 (en) 2015-10-29
US20170115279A1 (en) 2017-04-27
JP2017520237A (ja) 2017-07-27
US20200191776A1 (en) 2020-06-18
JP7348888B2 (ja) 2023-09-21
US20150301030A1 (en) 2015-10-22
US20160282327A1 (en) 2016-09-29
WO2015164367A1 (en) 2015-10-29
JP2017518738A (ja) 2017-07-13
US20150301029A1 (en) 2015-10-22
US9594075B2 (en) 2017-03-14
US20150301028A1 (en) 2015-10-22
EP3134728A1 (de) 2017-03-01
EP3134727A1 (de) 2017-03-01
CA2946514A1 (en) 2015-10-29
EP3134729A1 (de) 2017-03-01
CA2946385A1 (en) 2015-10-29
WO2015164378A1 (en) 2015-10-29
CA2946514C (en) 2023-08-29
US10613079B2 (en) 2020-04-07
WO2015164363A1 (en) 2015-10-29
US10107796B2 (en) 2018-10-23
JP2021006050A (ja) 2021-01-21
JP2017521044A (ja) 2017-08-03

Similar Documents

Publication Publication Date Title
US20200191776A1 (en) Diagnostic methods for neural disorders
US10161937B2 (en) Systems, methods, and workflows for optogenetics analysis
Nickolls et al. Transcriptional programming of human mechanosensory neuron subtypes from pluripotent stem cells
US20170292961A1 (en) Systems and methods for assessing inter-cell communication
EP3280797A1 (de) Erzeugung von funktionszellen aus stammzellen
US10048275B2 (en) Cardiotoxicity screening methods
US20220403331A1 (en) Methods for producing neural cells
Patani et al. Using human pluripotent stem cells to study post-transcriptional mechanisms of neurodegenerative diseases
Mok Morphology and Connectivity Impairments in Rett Syndrome Human Stem Cell-Derived Neurons
Laskary Magnetogenetic Stimulation of Human Embryonic Stem Cell-Derived Cardiomyocytes and Smooth Muscle Cells
Kaczynski Atoh7 functional specificity in the developing mammalian retina resides within its basic helix-loop-helix domain
Wilken Elucidating the cis-regulatory landscape of retinal development and regeneration
Ruby Genetic manipulation of Fezf2 in human embryonic stem cells and its application in studying central nervous system development and repair

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20161101

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
PUAG Search results despatched under rule 164(2) epc together with communication from examining division

Free format text: ORIGINAL CODE: 0009017

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20171205

B565 Issuance of search results under rule 164(2) epc

Effective date: 20171205

RIC1 Information provided on ipc code assigned before grant

Ipc: G01N 33/50 20060101AFI20171130BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20221005

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602015082956

Country of ref document: DE

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1557043

Country of ref document: AT

Kind code of ref document: T

Effective date: 20230415

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG9D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230629

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20230329

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1557043

Country of ref document: AT

Kind code of ref document: T

Effective date: 20230329

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230630

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230731

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20230928

Year of fee payment: 9

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230729

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20230929

Year of fee payment: 9

Ref country code: DE

Payment date: 20230928

Year of fee payment: 9

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230421

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602015082956

Country of ref document: DE

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20230430

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

26N No opposition filed

Effective date: 20240103

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230421

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230421

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230329